Activated T cells, nervous system-specific antigens and their uses

ABSTRACT

Compositions and methods to promote nerve regeneration or to confer neuroprotection and prevent or inhibit neuronal degeneration within the nervous system, eiteher the central nervous system or the peripheral nervous system, are provided. Treatment involves administering NS-specific activated T cells, or an NS-specific antigen or analog thereof, a peptide derived therefrom or an analog or derivative of said peptide, or a nucleotide sequence encoding said antigen or peptide, or any combination thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation-in-part of applicationSer. No. 09/314,161, filed May 19, 1999, which is a continuation-in-partof application No. PCT/US98/14715, filed Jul. 21, 1998, and is acontinuation-in-part of application Ser. No. 09/218,277, filed Dec. 22,1998, the entire contents of each of which are hereby incorporatedherein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to compositions and methods for thepromotion of nerve regeneration or prevention or inhibition of neuronaldegeneration to ameliorate the effects of injury or disease of thenervous system (NS). In the certain embodiments, activated T cells, anNS-specific antigen or peptide derived therefrom or a nucleotidesequence encoding an NS-specific antigen can be used to promote nerveregeneration or to confer neuroprotection and prevent or inhibitneuronal degeneration caused by injury or disease of nerves within thecentral nervous system or peripheral nervous system of a human subject.The compositions of the present invention may be administered alone ormay be optionally administered in any desired combination.

[0003] Abbreviations: APC: antigen-presenting cell; BSA: bovine serumalbumin; CAP: compound action potential; CFA: complete Freund'sadjuvant; CNS: central nervous system; 4-Di-10-Asp:4-(4-didecylamino)styryl)-N-methylpyridinium iodide; EAE: experimentalautoimmune encephalomyelitis; FCS: fetal calf serum; IFA: incompleteFreund's adjuvant MAG: myelin-associated glycoprotein; MBP: myelin basicprotein; MOG: myelin oligodendrocyte glycoprotein; NS: nervous system;OVA: ovalbumin; PBS: phosphate-buffered saline; PLP: proteolipid itprotein; PNS: peripheral nervous system; RGC: retinal ganglion cells;TCR: T-cell receptor.

BACKGROUND OF THE INVENTION

[0004] The nervous system comprises the central (CNS) and the peripheral(PNS) nervous system. The CNS is composed of the brain, the spinal cordand the visual system; the PNS consists of all of the other neuralelements, namely the nerves and ganglia outside of the brain and spinalcord.

[0005] Damage to the nervous system may result from a traumatic injury,such as penetrating trauma or blunt trauma, or a disease or disorderincluding, but not limited to Alzheimer's disease, Parkinson's disease,multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis(ALS), diabetic neuropathy, senile dementia, stroke and ischemia.

[0006] Maintenance of CNS integrity is a complex balancing act in whichcompromises are struck with the immune system. In most tissues, theimmune system plays an essential part in protection, repair, andhealing. In the CNS, because of its unique immune privilege,immunological reactions are relatively limited (Streilein, 1993). Agrowing body of evidence indicates that the failure of the mammalian CNSto achieve functional recovery after injury is a reflection of anineffective dialog between the damaged tissue and the immune system. Forexample, the restricted communication between the CNS and blood-bornemacrophages affects the capacity of axotomized axons to regrow;transplants of activated macrophages can promote CNS regrowth(Lazarov-Spiegler et al, 1996; Rapalino et al, 1998).

[0007] Activated T cells have been shown to enter the CNS parenchyma,irrespective of their antigen specificity, but only T cells capable ofreacting with a CNS antigen seem to persist there (Hickey et al, 1991;Werkele, 1993; Kramer et al, 1995). T cells reactive to antigens of CNSwhite matter, such as myelin basic protein (MBP), can induce theparalytic disease experimental autoimmune encephalomyelitis (EAE) withinseveral days of their inoculation into naive recipient rats (Ben-Nun etal, 1981a). Anti-MPB T cells may also be involved in the human diseasemultiple sclerosis (Ota et al, 1990; Martin, 1997). However, despitetheir pathogenic potential, anti-MBP T cell clones are present in theimmune systems of healthy subjects (Burns et al, 1983; Pette et al,1990; Martin et al, 1990; Schluesener et al, 1985). Activated T cells,which normally patrol the intact CNS, transiently accumulate at sites ofCNS white matter lesions (Hirschberg et al, 1998).

[0008] A catastrophic consequence of CNS injury is that the primarydamage is often compounded by the gradual secondary loss of adjacentneurons that apparently were undamaged, or only marginally damaged, bythe initial injury (Faden et al, 1992; Faden, 1993; McIntosh, 1993). Theprimary lesion causes changes in extracellular ion concentrations,elevation of amounts of free radicals, release of neurotransmitters,depletion of growth factors, and local inflammation These changestrigger a cascade of destructive events in the adjacent neurons thatinitially escaped the primary injury (Lynch et al, 1994; Bazan et al,1995; Wu et al, 1994). This secondary damage is mediated by activationof voltage-dependent or agonist-gated channels, ion leaks, activation ofcalcium-dependent enzymes such as proteases, lipases and nucleases,mitochondrial dysfunction and energy depletion, culminating in neuronalcell death (Yoshina et al, 1991; Hovda et al, 1991; Zivin et al, 1991;Yoles et al, 1992). The widespread loss of neurons beyond the losscaused directly by the primary injury has been called “secondarydegeneration.”

[0009] Another tragic consequence of CNS injury is that neurons in themammalian CNS do not undergo spontaneous regeneration following aninjury. Thus, a CNS injury causes permanent impairment of motor andsensory functions.

[0010] Spinal cord lesions, regardless of the severity of the injury,initially result in a complete functional paralysis known as spinalshock. Some spontaneous recovery from spinal shock may be observed,starting a few days after the injury and tapering off within three tofour weeks. The less severe the insult, the better the functionaloutcome. The extent of recovery is a function of the amount of undamagedtissue minus the loss due to secondary degeneration. Recovery frominjury would be improved by neuroprotective treatment that could reducesecondary degeneration.

[0011] The parent applications, application Ser. Nos. 09/218,277 and09/314,161 and PCT Publication WO 99/60021, describe the discovery madein the laboratory of the present inventors that activated T cells thatrecognize an antigen of the NS of the patient confer neuroprotection.More specifically, T cells reactive to MBP were shown to beneuroprotective in rat models of partially crushed optic nerve (see alsoMoalem et al, 1999a, the entire contents of which being herebyincorporated herein by reference) and of spinal cord injury (see alsoHauben et al, 2000, the entire contents of which being herebyincorporated herein by reference). Until recently, it had been thoughtthat immune cells do not participate in NS repair. Furthermore, anyimmune activity in the context of CNS damage was traditionallyconsidered detrimental for recovery. It was quite surprising to discoverthat NS-specific activated T cells could be used to protect nervoussystem tissue from secondary degeneration which may follow damage causedby injury or disease of the CNS or PNS. The mechanism of action of suchNS-specific T cells has yet to be discovered, but the massiveaccumulation of exogenously administered T cells at the site of CNSinjury suggests that the presence of T cells at the site of injury playsa prominent role in neuroprotection. It appears, however, that theaccumulation, though a necessary condition, is not sufficient for thepurpose, as T cells specific to the non-self antigen ovalbumin alsoaccumulate at the site, but have no neuroprotective effect (Hirschberget al, 1998).

[0012] In addition to the NS-specific activated T cells, theabove-referenced U.S. applications and PCT publication WO 99/60021disclose that therapy for amelioration of effects of injury or diseaseof NS can be carried out also with a natural or synthetic NS-specificantigen such as MAG, S-100, β-amyloid, Thy-1, P0, P2, a neurotransmitterreceptor, and preferably human MBP, human proteolipid protein (PLP), andhuman oligodendrocyte glycoprotein (MOG), or with a peptide derived froman NS-specific antigen such as a peptide comprising amino acids 51-70 ofMBP or amino acids 35-55 of MOG.

[0013] Citation or identification of any reference in this section orany other part of this application shall not be construed as anadmission that such reference is available as if prior art to theinvention.

SUMMARY OF THE INVENTION

[0014] The present invention is directed to methods and compositions forpromotion of nerve regeneration or for neuroprotection and prevention orinhibition of neuronal degeneration to ameliorate the effects of injuryto, or disease of, the nervous system (NS).

[0015] The present invention is based in part on the inventors'unexpected discovery that activated T cells that recognize an antigen ofthe NS of the patient promote nerve regeneration or conferneuroprotection. As used herein, “neuroprotection” refers to theprevention or inhibition of degenerative effects of injury or disease inthe NS. Since it was thought until recently that immune cells do notparticipate in nervous system repair, it was quite surprising todiscover that NS-specific activated T cells and also the NS-specificantigens themselves and peptides derived therefrom can be used topromote nerve regeneration or to protect nervous system tissue fromsecondary degeneration which may follow damage caused by injury ordisease of the CNS or PNS.

[0016] Thus, in one aspect, the invention relates to a method forpromoting nerve regeneration or for conferring neuroprotection andpreventing or inhibiting neuronal degeneration in the central nervoussystem or peripheral nervous system for ameliorating the effects ofinjury or disease, comprising administering to an individual in needthereof at least one ingredient selected from the group consisting of:

[0017] (a) NS-specific activated T cells;

[0018] (b) a NS-specific antigen or an analog thereof;

[0019] (c) a peptide derived from an NS-specific antigen or from ananalog thereof, or an analog or derivative of said peptide;

[0020] (d) a nucleotide sequence encoding an NS-specific antigen or ananalog thereof;

[0021] (e) a nucleotide sequence encoding a peptide derived from anNS-specific antigen or from an analog thereof, or an analog of saidpeptide; or

[0022] (f) any combination of (a)-(e).

[0023] In another aspect, the invention relates to a pharmaceuticalcomposition for promoting nerve regeneration or for neuroprotection andprevention or inhibition of neuronal degeneration in the CNS or PNS forameliorating the effects of injury or disease in the NS, comprising atherapeutically effective amount of at least one ingredient selectedfrom the group consisting of (a) to (e) above or any combination of(a)-(e).

[0024] The term “NS-specific antigen” as used herein refers to anantigen of the NS that specifically activates T cells such thatfollowing activation the activated T cells accumulate at a site ofinjury or disease in the NS of the patient. Examples of NS-specificantigens according to the invention include, but are not limited to,myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG),proteolipid protein (PLP), myelin-associated glycoprotein (MAG), S-100,β-amyloid, Thy-1, P0, P2, neurotransmitter receptors, the protein Nogo(Nogo-A, Nogo-B and Nogo-C) and the Nogo receptor (NgR). This definitionalso includes analogs of said NS-specific antigens as described in thesection on NS-specific antigens, analogs thereof, peptides derivedtherefrom and analogs and derivatives thereof of said peptideshereinafter.

BRIEF DESCRIPTION OF THE FIGURES

[0025]FIG. 1 is a bar graph showing the presence of T cells in uninjuredoptic nerve or in injured optic nerve one week after injury. Adult Lewisrats were injected with activated T cells of the anti-MBP (T_(MBP)),anti-OVA (T_(OVA)), anti-p277 (a peptide of the human hsp60) (Tp₂₇₇)lines, or with PBS, immediately after unilateral crush injury of theoptic nerve. Seven days later, both the injured and uninjured opticnerves were removed, cryosectioned and analyzed immunohistochemicallyfor the presence of immunolabeled T cells. T cells were counted at thesite of injury and at randomly selected areas in the uninjured opticnerves. The histogram shows the mean number of T cells per mm²±s.e.m.,counted in two to three sections of each nerve. Each group containedthree to four rats. The number of T cells was considerably higher ininjured nerves of rats injected with anti-MBP, anti-OVA or anti-p277 Tcells; statistical analysis (one-way ANOVA) showed significantdifferences between T cell numbers in injured optic nerves of ratsinjected with anti-MBP, anti-OVA, or anti-p277 T cells and the T cellnumbers in injured optic nerves of rats injected with PBS (P<0.001); andbetween injured optic nerves and uninjured optic nerves of rats injectedwith anti-MBP, anti-OVA, or anti-p277 T cells (P<0.001).

[0026]FIG. 2 is a bar graph illustrating that T cells specific to MBP,but not to OVA or p277, protect neurons from secondary degeneration.Immediately after optic nerve injury, rats were injected with anti-MBP,anti-OVA or anti-p277 T cells, or with PBS. The neurotracer dye4-Di-10-Asp was applied to optic nerves distal to the site of theinjury, immediately after injury (for assessment of primary damage) ortwo weeks later (for assessment of secondary degeneration). Five daysafter dye application, the retinas were excised and flat-mounted.Labeled retinal ganglion cells (RGCs) from three to five randomlyselected fields in each retina (all located at approximately the samedistance from the optic disk) were counted by fluorescence microscopy.RGC survival in each group of injured nerves was expressed as thepercentage of the total number of neurons spared after the primaryinjury (42% of neurons remained undamaged after the primary injury). Theneuroprotective effect of anti-MBP T cells compared with that of PBS wassignificant (P<0.001, one-way ANOVA). Anti-OVA T cells or anti-p277 Tcells did not differ significantly from PBS in their effects on theprotection of neurons that had escaped primary injury (P>0.05, one-wayANOVA). The results are a summary of five experiments. Each groupcontained five to ten rats.

[0027] FIGS. 3(A-C) present photomicrographs of retrogradely labeledretinas of injured optic nerves of rats. Immediately after unilateralcrush injury of their optic nerves, rats were injected with PBS (FIG.3A) or with activated anti-p277 T cells (FIG. 3B) or activated anti-MBPT cells (FIG. 3C). Two weeks later, the neurotracer dye 4-Di-10-Asp wasapplied to the optic nerves, distal to the site of injury. After 5 days,the retinas were excised and flat-mounted. Labeled (surviving) RGCs,located at approximately the same distance from the optic disk in eachretina, were photographed.

[0028] FIGS. 4(A-B) are graphs showing that clinical severity of EAE isnot influenced by an optic nerve crush injury. For the results presentedin FIG. 4A, Lewis rats, either uninjured (dash line) or immediatelyafter optic nerve crush injury (solid line), were injected withactivated anti-MBP T cells. EAE was evaluated according to aneurological paralysis scale. [Data points represent±s.e.m.] Theseresults represent a summary of three experiments. Each group containedfive to nine rats. FIG. 4B shows that the number of RGCs in theuninjured optic nerve is not influenced by injection of anti-MBP Tcells. Two weeks after the injection of anti-MBP T cells or PBS,4-Di-10-Asp was applied to the optic nerves. After 5 days the retinaswere excised and-flat-mounted. Labeled RGCs from five fields (located atapproximately the same distance from the optic disk) in each retina werecounted and the average number per mm² was calculated. There was nodifference between the numbers of labeled RGCs in rats injected withanti-MBP T cells (TMBP) and in PBS-injected control rats.

[0029]FIG. 5 is a bar graph showing that T cells specific to p51-70 ofMBP protect neurons from secondary degeneration. Immediately after opticnerve injury, rats were injected with anti-MBP T cells, anti-p51-70 Tcells, or PBS. 4-Di-10-Asp was applied to optic nerves distal to thesite of the injury, immediately after injury (for assessment of primarydamage) or two weeks later (for assessment of secondary degeneration).Five days after dye application, the retinas were excised andflat-mounted. Labeled RGCs from three to five randomly selected fieldsin each retina (all located at approximately the same distance from theoptic disk) were counted by fluorescence microscopy. RGC survival ineach group of injured nerves was expressed as the percentage of thetotal number of neurons spared after primary injury. Compared with thatof PBS treatment, the neuroprotective effects of anti-MBP and ofanti-p51-70 T cells were significant (P<0.001, one-way ANOVA).

[0030] FIGS. 6(A-B) are graphs showing that anti-MBP T cells increasethe compound action potential (CAP) amplitudes of injured optic nerves.Immediately after optic nerve injury, rats were injected with either PBSor activated anti-MBP T cells (T_(MBP)) . Two weeks later, the CAPs ofinjured (FIG. 6A) and uninjured (FIG. 6B) nerves were recorded. Therewere no significant differences in mean CAP amplitudes between uninjurednerves obtained from PBS-injected and anti-MBP T cell-injected rats(n-8; p=0.8, Student's t-test). The neuroprotective effect of anti-MBP Tcells (relative to PBS) on the injured nerve on day 14 after injury wassignificant (n=8, p=0.009, Student's t-test).

[0031] FIGS. 7(A-B) are graphs showing recovery of voluntary motoractivity as a function of time after contusion, with and withoutinjection of autoimmune anti-MBP T cells. (FIG. 7A) Twelve rats weredeeply anesthetized and laminectomized, and then subjected to acontusion insult produced by a 10 gram weight dropped from a height of50 mm. Six of the rats, selected at random, were then inoculated i.p.with 10⁷ anti-MBP T cells and the other six were inoculated with PBS. Atthe indicated time points, locomotor behavior in an open field wasscored by observers blinded to the treatment received by the rats.Results are expressed as the mean values for each group. The verticalbars indicate SEM. Differences tested by repeated ANOVA, including alltime points, were significant (p<0.05). (FIG. 7B) A similar experimentusing five PBS-treated animals and six animals treated with anti-MBP Tcells were all subjected to a more severe contusion. At the indicatedtime points, locomotor behavior in an open field was scored. The resultsare expressed as the mean values for each group. The vertical barsindicate SEM. Rats in the treated group are represented by open circlesand rats in the control group are represented by black circles.Horizontal bars show the median values. The inset shows the medianplateau values of the two groups.

[0032] FIGS. 8(A-C) show retrograde labeling of cell bodies at the rednucleus in rats treated with autoimmune anti-MBP T cells (8A) and incontrol injured (8B) rats. Three months after contusion and treatmentwith anti-MBP T cells, some rats from both the treated and the controlgroups were re-anesthetized and a dye was applied below the site of thecontusion. After five to seven days, the rats were again deeplyanesthetized and their brains were excised, processed, andcryosectioned. Sections taken through the red nucleus were inspected andanalyzed qualitatively and quantitatively under fluorescent and confocalmicroscopes. Significantly, more labeled nuclei were seen in the rednuclei of rats treated with anti-MBP T cells (8A) than in the red nucleiof PBS-treated rats (8B). The quantitative differences are shown in thebar graph (8C) and were obtained from animals with scores of 10 and 11in the T-cell-treated group and scores of 6 in the control group. Thebar graph shows mean±SD.

[0033]FIG. 9 is a series of photographs showing diffusion-weightedimaging of contused spinal cord treated with anti-MBP T cells. Spinalcords of MBP-T cell-treated and PBS-treated animals (with locomotionscores of 10 and 8, respectively) were excised under deep anesthesia,immediately fixed in 4% paraformaldehyde solution, and placed into 5 mmNMR tubes. Diffusion anisotropy was measured in a Bruker DMX 400widebore spectrometer using a microscopy probe with a 5-mm Helmholtzcoil and actively shielded magnetic field gradients. A multislice pulsedgradient spin echo experiment was performed with 9 axial slices, withthe central slice positioned at the center of the spinal injury. Imageswere acquired with TE of 31 ms, TR of 2000 ms, a diffusion time of 15ms, a diffusion gradient duration of 3 ms, field of view 0.6 mm, matrixsize 128×128, slice thickness 0.5 mm, and slice separation of 1.18 mm.Four diffusion gradient values of 0, 28, 49, and 71 g/cm were appliedalong the read direction (transverse diffusion) or along the slicedirection (longitudinal diffusion). Diffusion anisotropy is manifestedby increased signal intensity in the images with the highest transversediffusion gradient relative to the longitudinal diffusion gradient. Theexcised spinal cords of a PBS-treated rat and in the rat treated withMBP-T cells were subjected to diffusion-weighted MRI analysis. In thePBS-treated injured control, diffusion anisotropy was seen mainly insections near the proximal and distal stumps of the cord, with lowanisotropy in sections taken through the site of injury. In contrast, inthe treated rat, higher levels of diffusion anisotropy can be seen insections taken through the site of injury.

[0034]FIG. 10 is a graph illustrating inhibition of secondarydegeneration after optic nerve crush injury in adult rats. See text,Example 3, for experimental details. Rats were injected intradermallythrough the footpads with a 21-mer peptide based on MOG amino acidresidues 35-55 (MOG p35-55) ((50 μ/animal, chemically synthesized at theWeizmann Institute of Science, Rehovot, Israel) or PBS ten days prior tooptic nerve crush injury or MOG p35-55 in the absence of crush injury.MOG p35-55 was administered with IFA. Surviving optic nerve fibers weremonitored by retrograde labeling of RGCs. The number of RGCs in ratsinjected with PBS or MOG p35-55 was expressed as a percentage of thetotal number of neurons in rats injected with MOG p35-55 in the absenceof crush injury.

[0035]FIG. 11 is a graph illustrating inhibition in adult rats ofsecondary degeneration after optic nerve crush injury by MBP. See text,Example 4, for experimental details. MBP (Sigma, Israel) (1 mg in 0.5 mlsaline) was administered orally to adult rats by gavage using a bluntneedle. MBP was administered 5 times, i.e., every third day beginningtwo weeks prior to optic nerve crush injury. Surviving optic nervefibers were monitored by retrograde labeling of RGCs. The number of RGCsin treated rats was expressed as a percentage of the total number ofneurons in untreated rats following the injury.

[0036] FIGS. 12 (A-F) show expression of B7 co-stimulatory molecules inintact and injured rat optic nerve. Optic nerves were excised from adultLewis rats before (12A, 12B) and three days after injury (12C, 12D, 12E)and analyzed immunohistochemically for expression of the B7co-stimulatory molecule. The site of injury was delineated by GFAPstaining. Using calibrated cross-action forceps, the right optic nervewas subjected to a mild crush injury 1-2 mm from the eye. The uninjuredcointralateral nerve was left undisturbed. Immunohistochemical analysisof optic nerve antigens was performed as follows. Briefly, longitudinalcryosections of the excised nerves (20 μm thick) were picked up ontogelatin-coated glass and fixed with ethanol for ten minutes at roomtemperature. The sections were washed and incubated for one hour at roomtemperature with mouse monoclonal antibody to rat GFAP (BioMakor,Israel), diluted 1:100, and with antibodies to B7.2 co-stimulatorymolecule and the B7.1 co-stimulatory molecule (Pharmingen, San Diego,Calif.), diluted 1:25. The sections were washed again and incubated withrhodamine isothiocyanate-conjugated goat anti-mouse IgG (with minimalcross-reaction to rat, human, bovine and horse serum protein) go(Jackson ImmunoResearch, West Grove, Pa.), for one hour at room istemperature. All washing solutions contained PBS and 0.05% Tween-20. Alldiluting solutions contained PBS containing 3% fetal calf serum and 2%bovine serum albumin. The sections were treated with glycerol containing1,4-diazobicyclo-(2,2,2)-octane and were then viewed with a Zeissmicroscope. Note the morphological changes of the B7.2 positive cellsafter injury, from a rounded (12A, 12B) to a star-like shape (12C, 12D).The B7.2 positive cells were present at a higher density closer to theinjury site (12E). Expression of B7.1 was detectable only from day sevenand only at the injured site (12F).

[0037] FIGS. 13A-C show immunohistochemical analysis of T cells,macrophages or microglia, and B7.2 co-stimulatory molecules in theinjured optic nerves of rats fed MBP. Lewis rats aged 6-8 weeks were fed1 mg of bovine MBP (Sigma, Israel) (2 mg MBP/ml PBS) or 0.5 ml PBS onlyevery other day by gastric intubation using a stainless steel feedingneedle (Thomas Scientific, Swedesboro, N.J.) (Chen et al, 1994). Tendays after starting MBP, the right optic nerves were subjected tocalibrated crush injury, as described for FIG. 12. Three days later, thenerves were excised and prepared for immunohistochemical analysis of Tcells using mouse monoclonal antibodies to T cell receptor 11, diluted1:25, macrophages or microglia using anti-ED1 antibodies (Serotek,Oxford, U.K) diluted 1:250, astrocytes using anti-GFAP antibodies andB7.2 co-stimulatory molecules as described for FIG. 12. There were nosignificant quantitative differences in T cells or in ED-1 positivecells between injured optic nerves of PBS-fed (13A) and MBP-fed (13B)rats. The number of B7.2 positive cells at the site of injury of MBP-fedrats (13C) should be noted, as compared with injured controls (see FIG.12E above).

[0038]FIG. 14 is a graph showing the slowing of neuronal degeneration inrats with orally induced tolerance to MBP. Lewis rats were fed 1 mg MBPdaily, or every other day, or 4 times a day at two-hour intervals forfive consecutive days. Control animals were given PBS or the non-selfantigen OVA Sigma, Israel). Ten days after the start of MBP ingestion,the right optic nerves were subjected to a calibrated mild crush injury.Two weeks later the RGCs were retrogradely labelled by application ofthe fluorescent lipophilic dye 4-Di-10-Asp(Molecular Probes Europe BV,Netherlands), distally to the site of injury, as described. Briefly,complete axotomy was performed 1-2 mm from the distal border to theinjury site, and solid crystals (0.2-0.4 mm in diameter) of 4-Di-10-Aspwere immediately deposited at the site of the lesion. Retrogradelabeling of RGCs by the dye gives a reliable indication of the number ofstill-functioning neurons, as only intact axons can transport the dye totheir cell bodies in the retina. Six days after dye application, theretina was detached from the eye, prepared as a flattened whole mount in4% paraformaldehyde solution, and examined for labeled RGCs byfluorescence microscopy. RGCs were counted from three different regionsin the retina. The results are expressed as normalized percentage ofeach retina to untreated injured animal mean of the same experiment. Themedian of each group is shown as a bar (Control vs. MBP OTx4 ** P<0.01;Control vs. MBP OT ** P, 0.01; Control vs. OVA OT ns P>0.05.

[0039]FIG. 15 shows the nucleotide sequence of rat MBP gene, SEQ IDNO:1, Genbank accession number M25889 (Schaich et al, 1986).

[0040]FIG. 16 shows the nucleotide sequence of human MBP gene, SEQ IDNO:2, Genbank accession number M13577 (Kamholz et al, 1986).

[0041] FIGS. 17(A-F) show the nucleotide sequences of human PLP geneexons 1-7, SEQ ID NOs:3-8, respectively, Genbank accession numbersM15026-M15032, respectively (Diehl et al, 1986).

[0042]FIG. 18 shows the nucleotide sequence of human MOG gene, SEQ IDNO:9, Genbank accession number Z48051 (Roth et al, submitted (Jan. 17,1995) Roth, CNRS UPR 8291, CIGH, CHU Purpan, Toulouse, France, 31300;Gonzalez et al, 1996).

[0043]FIG. 19 shows the nucleotide sequence of rat PLP gene and variant,SEQ ID NO:10, Genbank accession number M16471 (Nave et al, 1987).

[0044]FIG. 20 shows the nucleotide sequence of rat MAG gene, SEQ IDNO:11, Genbank accession number M14871 (Arquint et al, 1987).

[0045]FIG. 21 shows the amino acid sequence of human MBP, SEQ ID NO:12,Genbank accession number 307160 (Kamholz et al, 1986).

[0046]FIG. 22 shows the amino acid sequence of human PLP, SEQ ID NO:13,Genbank accession number 387028.

[0047]FIG. 23 shows the amino acid sequence of human MOG, SEQ ID NO:14,Genbank accession number 793839 (Roth et al, 1995; Roth Submitted (JAN.17, 1995) Roth CNRS UPR 8291, CIGH, CHU Purpan, Toulouse, France, 31300;Gonzalez et al, 1996).

[0048] FIGS. 24(A-B) show that post-traumatic immunization with Nogopeptide p472 emulsified in CFA promotes functional recovery from spinalcord contusion in comparison to PBS+CFA-treated rats. Spinal cords ofmale SPD rats were laminectomized at the level of T9 and a 10-g rod wasdropped onto the laminectomized cord from a height of 50 mm (FIG. 24A)or of 25 mm (FIG. 24B). See text, Example 5, for experimental details.

[0049]FIG. 25 show that post-traumatic immunization with Nogo peptidep472 emulsified in CFA promotes functional recovery from spinal cordcontusion in comparison to PBS-treated or PBS+CFA-treated rats. Spinalcords of female SPD rats were laminectomized at the level of T9 and a10-g rod was dropped onto the laminectomized cord from a height of 50mm. See text, Example 5, for experimental details.

DETAILED DESCRIPTION OF THE INVENTION

[0050] As exposed above, the present invention relates to compositionsand methods for promoting nerve regeneration or for conferringneuroprotection and preventing or inhibiting neuronal degeneration inthe CNS or PNS for ameliorating the effects of injury or disease,comprising administering to an individual in need thereof at least oneingredient selected from the group consisting of:

[0051] (a) NS-specific activated T cells;

[0052] (b) a NS-specific antigen or an analog thereof;

[0053] (c) a peptide derived from an NS-specific antigen or from ananalog thereof, or an analog or derivative of said peptide;

[0054] (d) a nucleotide sequence encoding an NS-specific antigen or ananalog thereof;

[0055] (e) a nucleotide sequence encoding a peptide derived from anNS-specific antigen or from an analog thereof, or an analog of saidpeptide; or

[0056] (f) any combination of (a)-(e).

[0057] Merely for ease of explanation, the detailed description of thepresent invention is divided into the following sections: NS-specificactivated T cells and T-cell banks; NS-specific antigens, analogsthereof, peptides derived therefrom and analogs and derivatives thereofof said peptides; nucleotide sequences encoding NS-specific antigens,analogs thereof, peptides derived therefrom and analogs thereof;therapeutic uses; and formulations and modes of administration.

[0058] NS-Specific Activated T Cells and T-Cell Banks

[0059] In one embodiment of the invention, NS-specific activated T cellscan be used in an amount which is effective to confer neuroprotectionfor ameliorating or inhibiting the effects of injury or disease of theCNS or PNS that result in NS degeneration or for promoting regenerationin the NS, in particular the CNS, as described in the section ontherapeutic uses hereinafter.

[0060] In the practice of the invention, administration of NS-specificactivated T cells may optionally be in combination with an NS-specificantigen or an analog thereof or a peptide derived therefrom or an analogor derivative of said peptide. Additionally, oral administration ofNS-specific antigen or an analog thereof or a peptide derived therefromor an analog or derivative thereof, can be combined with activeimmunization to build up a critical T-cell response immediately afterinjury.

[0061] Activation of T cells is initiated by interaction of a TCRcomplex with a processed antigenic peptide bound to a MHC molecule onthe surface of an antigen-presenting cell (APC). As used herein, theterm “activated T cells” includes both (i) T cells that have beenactivated by exposure to a cognate antigen or peptide derived therefromor derivative thereof; and (ii) progeny of such activated T cells. Asused herein, a “cognate antigen” is an antigen that is specificallyrecognized by the TCR of a T cell that has been previously exposed tothe antigen. Alternatively, the T cell which has been previously exposedto the antigen may be activated by a mitogen, such as phytohemagglutinin(PHA) or concanavalin A (Con A).

[0062] The term “NS-specific activated T cell” as used herein refers toan activated T cell having specificity for an antigen of the NS, saidNS-specific antigen being an antigen of the NS that specificallyactivates T cells such that these activated T cells will accumulate at asite of injury or disease in the NS of the patient. The NS-specificantigen used to confer the specificity to the T cells may be a selfNS-antigen of the patient or a non-self NS-antigen of another individualor even of another species, or an analog of said NS-antigen, or apeptide derived from said NS-antigen or from said analog thereof, or ananalog or derivative of said peptide, all as described in the section onNS-specific antigens, analogs thereof, peptides derived therefrom andanalogs and derivatives thereof of said peptides hereinafter, as long asthe activated T cell recognizes an antigen in the NS of the patient.

[0063] If the disease being treated by the NS-specific activated T cellsof the invention is an autoimmune disease, in which the autoimmuneantigen is an NS antigen, the T cells which are used in accordance withthe present invention for the treatment of neural damage or degenerationcaused by such disease are preferably not activated against the sameautoimmune antigen involved in the disease. While the prior art hasdescribed methods of treating autoimmune diseases by administeringactivated T cells to create a tolerance to the autoimmune antigen, the Tcells of the present invention are not administered in such a way as tocreate tolerance, but are administered in such a way as to createaccumulation of the T cells at the site of injury or disease so as tofacilitate neural regeneration or to inhibit neural degeneration.

[0064] The prior art also discloses uses of immunotherapy againsttumors, including brain tumors, by administering T cells specific to anNS antigen in the tumor so that such T cells may induce an immune systemattack against the tumors. The present invention is not intended tocomprehend such prior art techniques. However, the present invention isintended to comprehend the inhibition of neural degeneration or theenhancement of neural regeneration in patients with brain tumors bymeans other than the prior art immunotherapy of brain tumors. Thus, forexample, NS-specific activated T cells, which are activated to anNS-antigen of the patient other than an antigen which is involved in thetumor, would be expected to be useful for the purpose of the presentinvention and would not have been suggested by known immunotherapytechniques.

[0065] The NS-specific activated T cells are preferably autologous, mostpreferably of the CD4 and/or CD8 phenotypes, but they may also besemi-allogeneic T cells or allogeneic T cells from related donors, e.g.,siblings, parents, children, or from donors with the same HLA type(HLA-matched) or a very similar HLA type (HLA-partially matched), oreven from unrelated donors.

[0066] Thus, in addition to the use of autologous T cells isolated fromthe subject, the present invention also comprehends the use ofsemi-allogeneic T cells for neuroprotection. The T cells may be preparedas short- or long-term lines and stored by conventional cryopreservationmethods for thawing and administration, either immediately or afterculturing for 1-3 days, to a subject suffering from injury to the CNSand in need of T-cell neuroprotection.

[0067] The use of semi-allogeneic T cells is based on the fact that Tcells can recognize a specific antigen epitope presented by foreign APC,provided that the APC expresses the MHC molecule, class I or class II,to which the specific responding T-cell population is restricted, alongwith the antigen epitope recognized by the T cells. Thus, asemi-allogeneic population of T cells that can recognize at least oneallelic product of the subject's MHC molecules, preferably a class IIHLA-DR or HLA-DQ or other HLA molecule, and that is specific for aNS-associated antigen epitope, will be able to recognize the NS antigenin the subject's area of NS damage and produce the neededneuro-protective effect. There is little or no polymorphism in theadhesion molecules, leukocyte migration molecules, and accessorymolecules needed for the T cells to migrate to the area of damage,accumulate there, and undergo activation. Thus, the semi-allogeneic Tcells will be able to migrate and accumulate at the CNS site in need ofneuroprotection and will be activated to produce the desired effect.

[0068] It is known that semi-allogeneic T cells will be rejected by thesubject's immune system, but that rejection requires about two weeks todevelop. Hence, the semi-allogeneic T cells will have the two-weekwindow of opportunity needed to exert neuroprotection. After two weeks,the semi-allogeneic T cells will be rejected from the body of thesubject, but that rejection is advantageous to the subject because itwill rid the subject of the foreign T cells and prevent any untowardconsequences of the activated T cells. The semi-allogeneic T cells thusprovide an important safety factor and are a preferred embodiment.

[0069] It is known that a relatively small number of HLA class IImolecules are shared by most individuals in a population. For example,about 50% of the Jewish population express the HLA-DR5 gene. Thus, abank of specific T cells reactive to NS-antigen epitopes that arerestricted to HLA-DR5 would be useful in 50% of that population. Theentire population can be covered essentially by a small number ofadditional T cell lines restricted to a few other prevalent HLAmolecules, such as DR1, DR4, DR2, etc. Thus, a functional bank ofuniform T cell lines can be prepared and stored for immediate use inalmost any individual in a given population. Such a bank of T cellswould overcome any technical problems in obtaining a sufficient numberof specific T cells from the subject in need of neuroprotection duringthe open window of treatment opportunity. The semi-allogeneic T cellswill be safely rejected after accomplishing their role ofneuroprotection. This aspect of the invention does not contradict, andis in addition to the use of autologous T cells as described herein.

[0070] The NS-specific activated T cells are preferably non-attenuated,although attenuated NS-specific activated T cells may be used. T cellsmay be attenuated using methods well-known in the art including, but notlimited to, by gamma-irradiation, e.g.,, 1.5-10.0 Rads (Ben-Nun et al,1981; Ben-Nun and Cohen, 1982); and/or by pressure treatment, forexample as described in U.S. Pat. No. 4,996,194 (Cohen et al); and/or bychemical cross-linking with an agent such as formaldehyde,glutaraldehyde and the like, for example as described in U.S. Pat. No.4,996,194 (Cohen et al); and/or by cross-linking and photoactivationwith light with a photoactivatable psoralen compound, for example asdescribed in U.S. Pat. No. 5,114,721 (Cohen et al); and/or by acytoskeletal disrupting agent such as cytochalsin and colchicine, forexample as described in U.S. Pat. No. 4,996,194 (Cohen et al). In apreferred embodiment the NS-specific activated T cells are isolated asdescribed below. T cells can be isolated and purified according tomethods known in the art (Mor and Cohen, 1995). For an illustrativeexample, see Example 1, Materials and Methods.

[0071] Circulating T cells of a subject which recognize an NS-antigenare isolated and expanded using known procedures (Burns et al, 1983;Pette et al, 1990; Martin et al, 1990; Schluesener et al, 1985;Suruhan-Dires Keneli et al, 1993, which are incorporated herein byreference in their entirety). In order to obtain NS-specific activated Tcells, T cells are isolated and the NS-specific activated T cells arethen expanded.

[0072] The isolated T cells may be activated by exposure of the cells toone or more of a variety of natural or synthetic NS-specific antigens orepitopes as described in section on NS-specific antigens, analogsthereof, peptides derived therefrom and analogs and derivatives thereofof said peptides hereinafter. During ex vivo activation of the T cells,the T cells may be activated by culturing in medium to which at leastone suitable growth promoting factor has been added, such as cytokines,e.g., TNF-α, IL-2 and/or IL-4.

[0073] In one embodiment, the NS-specific activated T cells endogenouslyproduce a substance that ameliorates the effects of injury or disease inthe NS.

[0074] In another embodiment, the NS-specific activated T cellsendogenously produce a substance that stimulates other cells, including,but not limited to, transforming growth factor-β (TGF-β), nerve growthfactor (NGF), neurotrophic factor 3(NT 3), neurotrophic factor 4/5(NT-4/5), brain derived neurotrophic factor (BDNF); IFN-γ and IL-6,wherein the other cells, directly or indirectly, ameliorate the effectsof injury or disease.

[0075] Following their proliferation in vitro, the T cells areadministered to a mammalian, preferably a human, subject. T cellexpansion is preferably performed using peptides corresponding tosequences in a non-pathogenic, NS-specific, self-protein.

[0076] A subject can initially be immunized with an NS-specific antigenusing a non-pathogenic peptide of the self-protein. A T-cell preparationcan be prepared from the blood of such immunized subjects, preferablyfrom T cells selected for their specificity towards the NS-specificantigen. The selected T cells can then be stimulated to produce a T cellline specific to the self-antigen (Ben-Nun and Cohen, 1982).

[0077] NS-specific antigen activated T cells, obtained as describedabove, can be used immediately or may be preserved for later use, e.g.,by cryopreservation as described below. NS-specific activated T cellsmay also be obtained using previously cryopreserved T cells, i.e., afterthawing the cells, the T cells may be incubated with NS-specificantigen, optimally together with thymocytes, to obtain a preparation ofNS-specific activated T cells.

[0078] As will be evident to those skilled in the art, the T cells canbe preserved, e.g., by cryopreservation, either before or after culture.

[0079] Cryopreservation agents which can be used include, but are notlimited to, dimethyl sulfoxide (DMSO) (Lovelock and Bishop, 1959;Ashwood-Smith, 1961), polyvinylpyrrolidone (Rinfret, 1960), glycerol,polyethylene glycol (Sloviter and Ravdin, 1962), albumin, dextran,sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol (Rowe etal, 1962), D-sorbitol, i-inositol, D-lactose, choline chloride (Benderet al, 1960), amino acids (Phan The Tran and Bender, 1960), methanol,acetamide, glycerol monoacetate (Lovelock, 1954), inorganic salts (PhanThe Tran and Bender, 1960 and 1961) and DMSO combined with hydroxyethylstarch and human serum albumin (Zaroulis and Leiderman, 1980).

[0080] A controlled cooling rate is critical. Different cryoprotectiveagents (Rapatz et al, 1968) and different cell types have differentoptimal cooling rates. See, e.g., Rowe and Rinfret, 1962; Rowe, 1966;Lewis et al, 1967; Mazur, 1970) for effects of cooling velocity onsurvival of cells and on their transplantation potential. The heat offusion phase where water turns to ice should be minimal. The coolingprocedure can be carried out by use of, e.g., a programmable freezingdevice or a methanol bath procedure.

[0081] Programmable freezing apparatuses allow determination of optimalcooling rates and facilitate standard reproducible cooling. Programmablecontrolled-rate freezers such as Cryomed or Planar permit tuning of thefreezing regimen to the desired cooling rate curve.

[0082] After thorough freezing, cells can be rapidly transferred to along-term cryogenic storage vessel. In one embodiment, samples can becryogenically stored in mechanical freezers, such as freezers thatmaintain a temperature of about −80° C. or about −20° C. In a preferredembodiment, samples can be cryogenically stored in liquid nitrogen(−196° C.) or its vapor. Such storage is greatly facilitated by theavailability of highly efficient liquid nitrogen refrigerators, whichresemble large Thermos containers with an extremely low vacuum andinternal super insulation, such that heat leakage and nitrogen lossesare kept to an absolute minimum.

[0083] Considerations and procedures for the manipulation,cryopreservation, and long term storage of T cells can be found, forexample, in the following references, incorporated by reference herein:Gorin, 1986; Bone-Marrow Conservation, it Culture and Transplantation,1968.

[0084] Other methods of cryopreservation of viable cells, ormodifications thereof, are available and envisioned for use, e.g., coldmetal-mirror techniques. See Livesey and Linner, 1987; Linner et al,1986; see also U.S. Pat. No. 4,199,022 by Senken et al, U.S. Pat. No.3,753,357 by Schwartz, U.S. Pat. No. 4,559,298 by Fahy.

[0085] Frozen cells are preferably thawed quickly (e.g., in a water bathmaintained at 37-47° C.) and chilled immediately upon thawing. It may bedesirable to treat the cells in order to prevent cellular clumping uponthawing. To prevent clumping, various procedures can be used, includingbut not limited to the addition before or after freezing of DNAse(Spitzer et al, 1980), low molecular weight dextran and citrate,citrate, hydroxyethyl starch (Stiff et al, 1983), or acid citratedextrose (Zaroulis and Leiderman, 1980), etc.

[0086] The cryoprotective agent, if toxic in humans, should be removedprior to therapeutic use of the thawed T cells. One way in which toremove the cryoprotective agent is by dilution to an insignificantconcentration.

[0087] Once frozen T cells have been thawed and recovered, they are usedto promote neuroprotection as described herein with respect tonon-frozen T cells. Once thawed, the T cells may be used immediately,assuming that they were activated prior to freezing. Preferably,however, the thawed cells are cultured before injection to the patientin order to eliminate non-viable cells. Furthermore, in the course ofthis culturing over a period of about one to three days, an appropriateactivating agent can be added so as to activate the cells, if the frozencells were resting T cells, or to help the cells achieve a higher rateof activation if they were activated prior to freezing. Usually, time isavailable to allow such a culturing step prior to administration as theT cells may be administered as long as a week after injury, and possiblylonger, and still maintain their neuro-regenerative and neuroprotectiveeffect.

[0088] To minimize secondary damage after nerve injury, patients can betreated by administering autologous or semi-allogeneic T lymphocytessensitized to at least one appropriate NS-antigen. As the window ofopportunity has not yet been precisely defined, therapy should beadministered as soon as possible after the primary injury to maximizethe chances of success, preferably within about one week.

[0089] To bridge the gap between the time required for activation andthe time needed for treatment, a bank with autologous, semi-allogeneicor allogeneic T cells can be established for future use.

[0090] Thus, in another embodiment, the invention provides cell banksthat can be established to store NS-sensitized T cells forneuroprotective treatment of individuals at a later time, as needed.

[0091] In one embodiment, autologous T cells may be obtained from anindividual and the cell bank will contain personal vaults of autologousT lymphocytes prepared for future use for neuroprotective therapyagainst secondary degeneration in case of NS injury. T lymphocytes areisolated from the blood, sensitized to a NS-antigen, and the cells arethen frozen and suitably stored under the person's name, identitynumber, and blood group, in a cell bank until needed.

[0092] Additionally, autologous stem cells of the CNS can be processedand stored for potential use by an individual patient in the event oftraumatic disorders of the NS such as ischemia or mechanical injury, aswell as for treating neurodegenerative conditions such as Alzheimer'sdisease or Parkinson's disease.

[0093] Alternatively, allogeneic or semi-allogeneic T cells may bestored such that a bank of T cells of each of the most common MHC-classII types are present. The semi-allogeneic or allogeneic T cells arestored frozen for use by any individual who shares one MHC type IImolecule with the source of the T cells.

[0094] In case an individual is to be treated for an injury, preferablyautologous stored T cells are used, but, if autologous T cells are notavailable, then cells should be used which share an MHC type II moleculewith the patient, and these would be expected to be operable in thatindividual.

[0095] The cells are preferably stored in an activated state afterexposure to an NS-antigen or peptide derived therefrom. However, thecells may also be stored in a resting state and activated once they arethawed and prepared for use. The cell lines of the bank are preferablycryopreserved. The cell lines are prepared in any way which is wellknown in the art. Once the cells are thawed, they are preferablycultured prior to injection in order to eliminate non-viable cells.During this culturing, the cells can be activated or reactivated usingthe same NS-antigen or peptide as used in the original activation.Alternatively, activation may be achieved by culturing in the presenceof a mitogen, such as phytohemagglutinin (PHA) or concanavalin A(preferably the former). This will place the cells into an even higherstate of activation. The few days that it takes to culture the cellsshould not be detrimental to the patient as the treatment in accordancewith the present invention may occuo still be effective. Alternatively,if time is of the essencer any time up to a week or more after theinjury in order t, the stored cells may be administered immediatelyafter thawing.

[0096] NS-Specific Antigens, Analogs thereof, Peptides DerivedTherefrom, and Analogs and Derivatives Thereof

[0097] The term “NS-specific antigen” as used herein refers to anantigen of the NS that specifically activates T cells such thatfollowing activation the activated T cells accumulate at a site ofinjury or disease in the NS of the patient.

[0098] The NS-specific antigen used according to the present inventionmay be an antigen obtained from NS tissue, preferably from tissue at asite of CNS injury or disease. It may be a crude NS-tissue preparation,e.g., derived from NS tissue obtained from mammalian NS that may includecells, both living or dead cells, membrane fractions of such cells ortissue, etc., and may be obtained by an NS biopsy or necropsy from amammal, preferably human, tissue including, but not limited to, from asite of CNS injury; from cadavers; and from cell lines grown in culture.

[0099] In one embodiment, the NS-specific antigen is an isolated orpurified antigen. The NS-specific antigen may be isolated and purifiedby standard methods including chromatography (e.g., ion exchange,affinity, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of antigens. The functional properties may be evaluatedusing any suitable assay. Additionally, an NS-specific antigen may be aprotein obtained by genetic engineering, chemically synthesized, etc.

[0100] In the practice of the invention, natural or syntheticNS-specific antigens are preferred and include, without being limitedto, myelin basic protein (MBP), proteolipid protein (PLP), myelinoligodendrocyte glycoprotein (MOG), myelin-associated glycoprotein(MAG), S-100, β-amyloid, Thy-1, P0, P2, neurotransmitter receptors, Nogoand Nogo receptor (NgR).

[0101] Specific illustrative examples of useful NS-specific antigensinclude but are not limited to, human MBP, depicted in FIG. 21 (SEQ IDNO:12); human PLP, depicted in FIG. 22 (SEQ ID NO:13); human MOG,depicted in FIG. 23 (SEQ ID NO:14), rat Nogo A, B and C (Chen et al,2000; WO 00/31235) (SEQ ID NOs:18, 20 and 21), peptide p472 (SEQ IDNO:19), human Nogo A, B and C (Prinjha et al, 2000) (SEQ ID NOs:23-25),and human or mouse Nogo receptor (NgR) (Fournier et al, 2001) (SEQ IDNOs:26 and 27, respectively).

[0102] Also encompassed by the present invention are analogs ofNS-specific antigens including, but not being limited to, thosemolecules comprising regions that are substantially homologous to thefull-length NS-specific antigen, or fragments thereof. In variousembodiments, these analogs will have at least 60% or 70% or 80% or 90%or 95% identity over an amino acid sequence of identical size or whencompared to an aligned sequence in which the alignment is done by acomputer homology program known in the art or whose encoding nucleicacid is capable of hybridizing to a coding nucleotide sequence of thefull-length NS-specific antigen, under high stringency, moderatestringency, or low stringency conditions. Computer programs fordetermining homology may include, but are not limited to, TBLASTN,BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988; Altschulet al, 1990; Thompson, et al, 1994; Higgins, et al, 1996).

[0103] The NS-specific antigen analogs of the invention can be producedby various methods known in the art. The manipulations which result intheir production can occur at the gene or protein level. For example, acloned gene sequence can be modified by any of numerous strategies knownin the art (Maniatis, 1990). The sequence can be cleaved at appropriatesites with restriction endonuclease(s), followed by further enzymaticmodification if desired, isolated, and ligated in vitro.

[0104] Additionally, the coding nucleic acid sequence can be mutated invitro or in vivo, to create and/or destroy translation, initiation,and/or termination sequences, or to create variations in coding regionsand/or form new restriction endonuclease sites or destroy preexistingones, to facilitate further in vitro modification. Any technique formutagenesis known in the art can be used, including but not limited to,chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson, etal, 1978), etc.

[0105] Manipulations may also be made at the protein level. Includedwithin the scope of the invention are NS-specific antigen derivativeswhich are differentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Any of numerouschemical modifications may be carried out by known techniques, includingbut not limited to specific chemical cleavage by cyanogen bromide,trypsin, chymotrypsin, papain, V8 protease, NaBH₄; acetylation,formylation, oxidation, reduction; metabolic synthesis in the presenceof tunicamycin; etc.

[0106] In a preferred embodiment, the invention relates to peptidesderived from NS-specific antigens or from analogs thereof and to analogsor derivatives of said peptides, which are functionally active, i.e.,they are capable of displaying one or more known functional activitiesassociated with a full-length NS-specific antigen. Such functionalactivities include, but are not limited to, antigenicity (ability tobind, or compete with an NS-antigen for binding, to an anti-NS-specificantibody), immunogenicity (ability to generate antibody which binds toan NS-specific protein), and ability to interact with T cells, resultingin activation comparable to that obtained using the correspondingfull-length NS-specific antigen. The crucial test is that the antigenwhich is used for activating the T cells causes the T cells to becapable of recognizing an antigen in the NS of the mammal (patient)being treated.

[0107] The NS-antigen derived peptide may be either: (1) an immunogenicpeptide, i.e., a peptide that can elicit a human T-cell responsedetected by a T-cell proliferation assay or by cytokine, e.g., IFN-γ,IL-2, IL-4 or IL-10, production, or (2) a “cryptic epitope” (alsodesignated herein as “immunosilent” or “non-immunodominant” epitope),i.e., a peptide that by itself can induce a T-cell immune response thatis not induced by the whole antigen protein (see Moalem et al, 1999).

[0108] A peptide derived from a NS-specific antigen preferably has asequence comprised within the NS-specific antigen sequence and has atleast 10, 13, 15, 18, 20 or 50 contiguous amino acids of the NS-specificantigen sequence. In one embodiment, the peptide derived from anNS-specific antigen is a “cryptic epitope” of the antigen. A crypticepitope activates specific T cells after an animal is immunized with theparticular peptide, but not with the whole antigen. Cryptic epitopes foruse in the present invention include, but are not limited to, peptidesof the MBP sequence: peptides p11-30, p51-70, p87-99, p91-110, p131-150,and p151-170. Such cryptic epitopes are particularly preferred as Tcells activated thereby will accumulate at the injury site, but areparticularly weak in autoimmunity. Thus, they would be expected to havefewer side effects.

[0109] In another embodiment, the peptide derived from an NS-specificantigen is an immunogenic epitope of the antigen.

[0110] Examples of further peptides according to the invention areimmunogenic peptides derived from the Nogo protein sequence such as, butnot being limited to, the 18-mer p472 Nogo peptide (SEQ ID NO:18) andpeptides derived from the Nogo receptor (Fournier et al, 2001) such asthe 15-mer peptides of the sequences:

[0111] S G V P S N L P Q R L A G R D (SEQ ID NO:28)

[0112] T R S H C R L G Q A G S G S S (SEQ ID NO:29)

[0113] In still another embodiment of the invention, the peptide is ananalog of a peptide derived from an NS-specific antigen that isimmunogenic but not encephalitogenic. The most suitable peptides forthis purpose are those in which an encephalitogenic self-peptide ismodified at the T-cell receptor (TCR) binding site and not at the MHCbinding site(s), so that the immune response is activated but notanergized (Karin et al, 1998; Vergelli et al, 1996).

[0114] These analogs, also referred herein as modified peptides oraltered peptides, may be produced by replacement of one or more aminoacid residues of the peptide by other amino acid residues, preferably intheir TCR binding site. Suitable replacements are those in which chargedamino residues like lysine, proline or arginine are replaced by glycineor alanine residues. For example, altered peptides can be produced frompeptides p11-30, p51-70, p87-99, p91-110, p131-150, and p151-170 ofhuman MBP, for example from the p87-99 peptide in which the lysine 91 isreplaced by glycine and/or the proline 96 is replaced by an alanineresidue, thus converting an encephalitogenic peptide in immunogenic butnon-encephalitogenic peptide that still recognizes the TCR. In the sameway, altered peptides can be produced from the encephalitogenic p472Nogo peptide (Nogo p623-640) by replacement of the lys 628 residue andfrom the Nogo receptor peptides above by replacement of the arg (R)residue by Val or Ala or another similar residue.

[0115] In addition, the analogs also comprise replacement of one or moreamino acid residues of the peptide or addition to the peptide ofnon-natural amino acids including, but not limited to, the D-isomers ofthe common amino acids, α-aminoisobutyric acid; 4-aminobutyric acid(Abu); 2-Abu (γ-Abu); 6-amino hexanoic acid (ε-Ahx); 2-aminoisobutyricacid (Aib); 3-aminopropionic acid; ornithine; norleucine (Nle);norvaline (Nva); hydroxyproline; sarcosine; citrulline; cysteic acid;t-butylglycine; t-butylalanine; phenylgylcine; cyclohexylalanine;β-alanine; fluoro-amino acids; designer amino acids such as β-methylamino acids, Cα-methyl amino acids, Nα-methyl amino acids, and aminoacid analogs in general. Furthermore, the amino acid can be D(dextrorotary) or L (levorotary).

[0116] Furthermore, the invention also comprises chemical derivatives ofthe peptides of the invention including, but not being limited to,esters of both carboxylic and hydroxy groups, amides, and the like.

[0117] The NS-specific antigen peptides of the invention can bechemically synthesized- For example, a peptide corresponding to aportion of an antigen which comprises the desired domain or whichmediates the desired activity can be synthesized by use of a peptidesynthesizer.

[0118] The functional activity of NS-specific antigens and peptidesderived therefrom and analogs and derivatives thereof can be assayed byvarious methods known in the art, including, but not limited to, T-cellproliferation assays (Mor and Cohen, 1995) and cytokine productionassays.

[0119] An NS-specific antigen or peptide derived therefrom or derivativethereof may be kept in solution or may be provided in a dry form, e.g.,as a powder or lyophilizate, to be mixed with appropriate solution priorto use. They may be used both as ingredients of pharmaceuticalcompositions for neuroprotection and preventing or inhibiting theeffects of injury or disease that result in NS degeneration or forpromoting nerve regeneration in the NS, particularly in the CNS as wellas for in vivo or in vitro activation of T cells.

[0120] Nucleotide Sequences Encoding NS-Antigens and Peptides DerivedTherefrom

[0121] The present invention further provides pharmaceuticalcompositions comprising a therapeutically effective amount of anucleotide sequence encoding an NS-specific antigen or a peptide derivedtherefrom or an analog thereof and methods of use of such compositionsto promote nerve regeneration or for neuroprotection and prevention orinhibition of neuronal degeneration in the CNS or PNS in which theamount is effective to ameliorate the effects of an injury or disease ofthe NS.

[0122] Specific illustrative examples of useful nucleotide sequencesencoding NS-specific antigens or peptides derived from an NS-specificantigen include, but are not limited to, nucleotide sequences encodingrat MBP, depicted in FIG. 15 (SEQ ID NO:1); human MBP, depicted in FIG.16 (SEQ ID NO:2); human PLP, depicted in FIGS. 17(A-F) (SEQ ID NOs:3-8);human MOG, depicted in FIG. 18 (SEQ ID NO:9); rat PLP and variant,depicted in FIG. 19 (SEQ ID NO:10); rat MAG, depicted in FIG. 20 (SEQ IDNO:11); rat Nogo (SEQ ID NO:17); and human Nogo (SEQ ID NO:22). Otherillustrative examples are the nucleotide sequences disclosed in Chen etal (2000), Prinjha et al (2000) and Fournier et al (2001) (the contentsof each of which being hereby incorporated herein by reference) encodingrat and human Noga A, B and C and mouse and human NgR.

[0123] Therapeutic Uses

[0124] The T cells, NS-specific antigens, analogs thereof, peptidesderived therefrom and analogs and derivatives thereof, and nucleotidesequences described in the previous sections and compositions comprisingthem may be used to promote nerve regeneration or to conferneuroprotection and prevent or inhibit secondary degeneration which mayotherwise follow primary NS injury, e.g., spinal cord injury, blunttrauma, penetrating trauma, hemorrhagic stroke, ischemic stroke ordamages caused by surgery such as tumor excision.

[0125] In addition, such compositions may be used to ameliorate theeffects of disease that result in a degenerative process, e.g.,degeneration occurring in either gray or white matter (or both) as aresult of various diseases or disorders, including, without limitation:diabetic neuropathy, senile dementias, Alzheimer's disease, Parkinson'sdisease, facial nerve (Bell's) palsy, glaucoma, Huntington's chorea,amyotrophic lateral sclerosis (ALS), non-arteritic optic neuropathy,intervertebral disc herniation, vitamin deficiency, prion diseases suchas Creutzfeldt-Jakob disease, carpal tunnel syndrome, peripheralneuropathies associated with various diseases, including but not limitedto, uremia, porphyria, hypoglycemia, Sjorgren Larsson syndrome, acutesensory neuropathy, chronic ataxic neuropathy, biliary cirrhosis,primary amyloidosis, obstructive lung diseases, acromegaly,malabsorption syndromes, polycythemia vera, IgA- and IgG gamma-pathies,complications of various drugs (e.g., metronidazole) and toxins (e.g.,alcohol or organophosphates), Charcot-Marie-Tooth disease, ataxiatelangectasia, Friedreich's ataxia, amyloid polyneuropathies,adrenomyeloneuropathy, Giant axonal neuropathy, Refsum's disease,Fabry's disease, lipoproteinemia, etc.

[0126] In a preferred embodiment, the NS-specific activated T cells, theNS-specific antigens, peptides derived therefrom, analogs andderivatives thereof or the nucleotides encoding said antigens, orpeptides or any combination thereof of the present invention are used totreat diseases or disorders where promotion of nerve regeneration orprevention or inhibition of secondary neural degeneration is indicated,which are not autoimmune diseases or neoplasias. In a preferredembodiment, the compositions of the present invention are administeredto a human subject.

[0127] While activated NS-specific T cells may have been used in theprior art in the course of treatment to develop tolerance to autoimmuneantigens in the treatment of autoimmune diseases, or in the course ofimmunotherapy in the treatment of NS neoplasms, the present inventioncan also be used to ameliorate the degenerative process caused byautoimmune diseases or neoplasms as long as it is used in a manner notsuggested by such prior art methods. Thus, for example, T cellsactivated by an autoimmune antigen have been suggested for use to createtolerance to the autoimmune antigen and, thus, ameliorate the autoimmunedisease. Such treatment, however, would not have suggested the use of Tcells directed to other NS antigens or NS antigens which will not inducetolerance to the autoimmune antigen or T cells which are administered insuch a way as to avoid creation of tolerance. Similarly, for neoplasms,the effects of the present invention can be obtained without usingimmunotherapy processes suggested in the prior art by, for example,using an NS antigen which does not appear in the neoplasm. T cellsactivated with such an antigen will still accumulate at the site ofneural degeneration and facilitate inhibition of this degeneration, eventhough it will not serve as immunotherapy for the tumor per se.

[0128] Nogo protein or a fragment thereof which are active in inhibitingcell proliferation have been disclosed as useful for treatment of aneoplastic disease of the CNS such as glioma, glioblastoma,medulloblastoma, craniopharyngioma, ependyoma, neuroblastoma andretinoblastoma. The present invention does not encompass the use of Nogoor a peptide derived therefrom for treatment of neoplasias in general,and for treatment of a neoplastic disease of the CNS, in particular.

[0129] Formulations and Administration

[0130] The present invention also provides pharmaceutical compositionsuseful in methods to promote nerve regeneration or to conferneuroprotection and prevent or inhibit neuronal degeneration in the CNSor PNS, comprising a therapeutically effective amount of at least oneingredient selected from the group consisting of:

[0131] (a) NS-specific activated T cells;

[0132] (b) a NS-specific antigen or an analog thereof;

[0133] (c) a peptide derived from an NS-specific antigen or from ananalog thereof, or an analog or derivative of said peptide;

[0134] (d) a nucleotide sequence encoding an NS-specific antigen or ananalog thereof;

[0135] (e) a nucleotide sequence encoding a peptide derived from anNS-specific antigen or from an analog thereof, or an analog of saidpeptide; or

[0136] (f) any combination of (a)-(e).

[0137] The compositions comprising ingredients (b) and/or (c) above arealso effective to activate T cells in vitro, wherein the activated Tcells inhibit or ameliorate the effects of an injury or disease of theNS.

[0138] Pharmaceutical compositions for use in accordance with thepresent invention may be formulated in conventional manner using one ormore physiologically acceptable carriers or excipients. The carrier(s)must be “acceptable” in the sense of being compatible with the otheringredients of the composition and not deleterious to the recipientthereof.

[0139] The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the therapeutic is administered. The carriers in thepharmaceutical composition may comprise a binder, such asmicrocrystalline cellulose, polyvinylpyrrolidone (polyvidone orpovidone), gum tragacanth, gelatin, starch, lactose or lactosemonochydrate; a disintegrating agent, such as alginic acid, maize starchand the like; a lubricant or surfactant, such as magnesium stearate orsodium lauryl sulphate; a glidant, such as colloidal silicon dioxide; asweetening agent, such as sucrose or saccharin; and/or a flavoringagent, such as peppermint, methyl salicylate, or orange flavoring.

[0140] Methods of administration include, but are not limited to,parenteral, e.g., intravenous, intraperitoneal, intramuscular,subcutaneous, mucosal (e.g., oral, intranasal, buccal, vaginal, rectal,intraocular), intrathecal, topical and intradermal routes.Administration can be systemic or local.

[0141] For oral administration, the pharmaceutical preparation may be inliquid form, for example, solutions, syrups or suspensions, or may bepresented as a drug product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalcompositions may take the form of, for example, tablets, lozenges orcapsules prepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell-known in the art.

[0142] Preparations for oral administration may be also suitablyformulated to give controlled release of the active compound.

[0143] The compositions may be formulated for parenteral administrationby injection, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers, with an added preservative. Thecompositions may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

[0144] The compositions may also be formulated as rectal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0145] For administration by inhalation, the compositions for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin, for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

[0146] In one embodiment, compositions comprising NS-specific activatedT cells, an NS-specific antigen or peptide derived therefrom, orderivative thereof, or a nucleotide sequence encoding such antigen orpeptide, are formulated in accordance with routine procedures aspharmaceutical compositions adapted for intravenous or intraperitonealadministration to human beings. Typically, compositions for intravenousadministration are solutions in sterile isotonic aqueous buffer. Wherenecessary, the composition may also include a solubilizing agent and alocal anesthetic such as lignocaine to ease pain at the site of theinjection. Generally, the ingredients are supplied either separately ormixed together. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water or saline forinjection can be provided so that the ingredients may be mixed prior toadministration.

[0147] Pharmaceutical compositions comprising NS-specific antigen orpeptide derived therefrom or derivative thereof may optionally beadministered with an adjuvant.

[0148] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.

[0149] In a preferred embodiment, the pharmaceutical compositions of theinvention are administered to a mammal, preferably a human, shortlyafter injury or detection of a degenerative lesion in the NS. Thetherapeutic methods of the invention may comprise administration of anNS-specific activated T cell or an NS-specific antigen or peptidederived therefrom or derivative thereof, or a nucleotide sequenceencoding such antigen or peptide, or any combination thereof. When usingcombination therapy, the NS-specific antigen may be administered before,concurrently or after administration of NS-specific activated T cells, apeptide derived from an NS-specific antigen or derivative thereof or anucleotide sequence encoding such antigen or peptide.

[0150] In one embodiment, the compositions of the invention areadministered in combination with one or more of the following: (a)mononuclear phagocytes, preferably cultured monocytes (as described inPCT publication No. WO 97/09985, which is incorporated herein byreference in its entirety), that have been stimulated to enhance theircapacity to promote neuronal regeneration; (b) a neurotrophic factorsuch as acidic fibroblast growth factor; and (c) an anti-inflammatorytherapeutic substance, e.g., an anti-inflammatory steroid, such asdexamethasone or methyl-prednisolone, or a non-steroidalanti-inflammatory peptide, such as Thr-Lys-Pro (TKP)).

[0151] In another embodiment, mononuclear phagocyte cells according toPCT Publication No. WO 97/09985 and U.S. patent application Ser. No.09/041,280, filed Mar. 11, 1998, are injected into the site of injury orlesion within the CNS, either concurrently, prior to, or followingparenteral administration of NS-specific activated T cells, anNS-specific antigen or peptide derived therefrom or derivative thereof,or a nucleotide sequence encoding such antigen or peptide

[0152] In another embodiment, administration of NS-specific activated Tcells, NS-specific antigen or peptide sequence encoding such antigen orpeptide, may be administered as a single dose or may be repeated,preferably at 2-week intervals and then at successively longer intervalsonce a month, once a quarter, once every six months, etc. The course oftreatment may last several months, several years or occasionally alsothrough the life-time of the individual, depending on the condition ordisease which is being treated. In the case of a CNS injury, thetreatment may range between several days to months or even years, untilthe condition has stabilized and there is no or only a limited risk ofdevelopment of secondary degeneration. In chronic human disease orParkinson's disease, the therapeutic treatment in accordance with theinvention may be for life.

[0153] As will be evident to those skilled in the art, the therapeuticeffect depends at times on the condition or disease to be treated, onthe individual's age and health condition, on other physical parameters(e.g., gender, weight, etc.) of the individual, as well as on variousother factors, e.g., whether the individual is taking other drugs, etc.

[0154] The optimal dose of the therapeutic compositions comprisingNS-specific activated T cells of the invention is proportional to thenumber of nerve fibers affected by NS injury or disease at the sitebeing treated. In a preferred embodiment, the dose ranges from about5×10⁶ to about 10⁷ for treating a lesion affecting about 10 nervefibers, such as a complete transection of a rat optic nerve, and rangesfrom about 10⁷ to about 10⁸ for treating a lesion affecting about10⁶-10⁷ nerve fibers, such as a complete transection of a human opticnerve. As will be evident to those skilled in the art, the dose of Tcells can be scaled up or down in proportion to the number of nervefibers thought to be affected at the lesion or site of injury beingtreated.

[0155] The following examples illustrate certain features of the presentinvention but are not intended to limit the scope of the presentinvention.

EXAMPLE 1

[0156] Accumulation of Activated T Cells in Injured Optic Nerve

Materials and Methods

[0157] Animals

[0158] Female Lewis rats were supplied by the Animal Breeding Center ofthe Weizmann Institute of Science (Rehovot, Israel), matched for age(8-12 weeks) and housed four to a cage in a light andtemperature-controlled room.

[0159] Media

[0160] The T-cell proliferation medium contained the following:Dulbecco's modified Eagle's medium (DMEM, Biological Industries, Israel)supplemented with 2 mM L-glutamine (L-Glu, Sigma, USA), 5×10⁻⁵ M2-mercaptoethanol (2-ME, Sigma), penicillin (100 IU/ml; BiologicalIndustries), streptomycin (100 μ/ml; Biological Industries), sodiumpyruvate (1 mM; Biological Industries), non-essential amino acids (1ml/100 ml; Biological Industries) and autologous rat serum 1% (vol/vol)(Mor et al, 1990). Propagation medium contained: DMEM, 2-ME, L-Glu,sodium pyruvate, non-essential amino acids and antibiotics in the sameconcentration as above with the addition of 10% fetal calf serum (FCS),and 10% T cell growth factor (TCGF) obtained from the supernatant ofconcanavalin A-stimulated spleen cells (Mor et al, 1990).

[0161] Antigens

[0162] MBP from the spinal cords of guinea pigs was prepared asdescribed (Hirshfeld, et al, 1970). OVA was purchased from Sigma (St.Louis, Mo.). The p51-70 of the rat 18.5 kDa isoform of MBP (sequence:APKRGSGKDSHTRTTHYG) (SEQ ID NO:15) and the p277 peptide of the humanhsp60 (sequence: VLGGGCALLRCPALDSLTPANED) (SEQ ID NO:16) (Elias et al,1991) were synthesized using the 9-fluorenylmethoxycarbonyl (Fmoc)technique with an automatic multiple peptide synthesizer (AMS 422,ABIMED, Langenfeld, Germany). The purity of the peptides was analyzed byHPLC and amino acid composition.

[0163] T Cell Lines

[0164] T-cell lines were generated from draining lymph node cellsobtained from Lewis rats immunized with an antigen (described above inAntigens). The antigen was dissolved in PBS (1 mg/ml) and emulsifiedwith an equal volume of IFA (Difco Laboratories, Detroit, Mich.)supplemented with 4 mg/ml Mycobacterium tuberculosis (Difco 15Laboratories, Detroit, Mich.). The emulsion (0.1 ml) was injected intohind foot pads of the rats. Ten days after the antigen was injected, therats were killed and draining lymph nodes were surgically removed anddissociated. The cells were washed and activated with the antigen (10μg/ml) in proliferation medium (described above in Media). Afterincubation for 72 h at 37° C., 90% relative humidity and 7% CO₂, thecells were transferred to propagation medium (described above in Media).Cells were grown in propagation medium for 4-10 days before beingre-exposed to antigen (10 μg/ml) in the presence of irradiated (2000rad) thymus cells (10⁷ cells/ml) in proliferation medium. The T celllines were expanded by repeated re-exposure and propagation.

[0165] Crush Injury of Rat Optic Nerve

[0166] Crush injury of the optic nerve was performed as previouslydescribed (Duvdevani et al, 1990). Briefly, rats were deeplyanesthetized by i.p. injection of Rompum (xylazine, 10 mg/kg; Vitamed,Israel) and Vetaler (ketamine, 50 mg/kg; Fort Dodge Laboratories, FortDodge, Iowa). Using a binocular operating microscope, a lateralcanthotomy was performed in the right eye and the conjunctiva wasincised lateral to the cornea. After separation of the retractor bulbimuscles, the optic nerve was exposed intraorbitally by blunt dissection.Using calibrated cross-action forceps, a moderate crush injury wasinflicted on the optic nerve, 2 mm form the eye (Duvdevani et al, 1990).The contralateral nerve was left undisturbed and was used as a control.

[0167] Immunocytochemistry of T Cells

[0168] Longitudinal cryostat nerve sections (20 μm thick) were picked uponto gelatin glass slides and frozen until preparation for fluorescentstaining. Sections were thawed and fixed in ethanol for 10 minutes atroom temperature, washed twice with double-distilled water (ddH₂O), andincubated for 3 minutes in PBS containing 0.05% polyoxyethylene-sorbitanmonolaurate (Tween-20; Sigma, USA). Sections were then incubated for 1hr at room temperature with a mouse monoclonal antibody directed againstrat T cell receptor (TCR) (1:100, Hunig et al, 1989), in PBS containing3% FCS and 2% BSA. After three washes with PBS containing 0.05%Tween-20, the sections were incubated with fluoresceinisothiocyanate-conjugated goat anti-mouse IgG (with minimalcross-section to rat, human, bovine and horse serum proteins) (JacksonImmunoResearcch, West Grove, Pa.) for one hour at room temperature. Thesections were then washed with PBS containing Tween-20 and treated withglycerol containing 1,4-diazobicyclo-(2,2,2) octane (Sigma), to inhibitquenching of fluorescence. The sections were viewed with a Zeismicroscope and cells were counted. Staining in the absence of firstantibody was negative.

Results

[0169]FIG. 1 shows accumulation of T cells measuredimmunohistochemically. The number of T cells was considerably higher ininjured nerves rats injected with anti-MBP, anti-OVA or anti-p277 cells;statistical analysis (one-way ANOVA) showed significant differencesbetween T cell numbers in injured optic nerves of rats injected withanti-MBP, anti-OVA, or anti-p277 T cells and in injured optic nerves ofrats injected with PBS (P<0.001); and between injured optic nerves anduninjured optic nerves of rats injected with anti-MBP, anti-OVA, oranti-p277 T cells (P<0.001).

EXAMPLE 2

[0170] Neuroprotection by Autoimmune Anti-MBP T Cells Material andMethods

[0171] Animals, media, antigens, crush injury of rat optic nerve,sectioning of nerves, T cell lines, and immunolabeling of nerve sectionsare described in Example 1, supra.

[0172] Retrograde Labeling and Measurement of Primary Damage andSecondary Degeneration

[0173] Primary damage of the optic nerve axons and their attached RGCswere measured after the immediate post-injury application of thefluorescent lipophilic dye 4-Di-10-Asp) (Molecular Probes Europe BV,Netherlands) distal to the site of injury. Only axons that are intactare capable of transporting the dye back to their cell bodies;therefore, the number of labeled cell bodies is a measure of the numberof axons that survived the primary damage. Secondary degeneration wasalso measured by application of the dye distal to the injury site, buttwo weeks after the primary lesion was inflicted. Application of theneurotracer dye distal to the site of the primary crush after two weeksensures that only axons that survived both the primary damage and thesecondary degeneration will be counted. This approach makes it possibleto differentiate between neurons that are still functionally intact andneurons in which the axons are injured but the cell bodies are stillviable, as only those neurons whose fibers are morphologically intactcan take up dye applied distally to the site of injury and transport itto their cell bodies. Using this method, the number of labeled RGCsreliably reflects the number of still functioning neurons. Labeling andmeasurement were done by exposing the right optic nerve for a secondtime, again without damaging the retinal blood supply. Complete axotomywas done 1-2 mm from the distal border of the injury site and solidcrystals (0.2-0.4 mm in diameter) of 4-Di-10-Asp were deposited at thesite of the newly formed axotomy. Uninjured optic nerves were similarlylabeled at approximately the same distance from the globe. Five daysafter dye application, the rats were killed. The retina was detachedfrom the eye, prepared as a flattened whole mount in 4% paraformaldehydesolution and examined for labeled RGCs by fluorescence microscopy. Thepercentage of RGCs surviving secondary degeneration was calculated usingthe following formula:

(Number of spared neurons after secondary degeneration)/(Number ofspared neurons after primary damage)×100.

[0174] Electrophysiological Recordings

[0175] Nerves were excised and their compound action potentials (CAPs)were recorded in vitro using a suction electrode experimental set-up(Yoles et al, 1996). At different times after injury and injection of Tcells or PBS, rats were killed by intraperitoneal injection ofpentobarbitone (170 mg/kg) (CTS Chemical Industries, Israel). Both opticnerves were removed while still attached to the optic chiasma, and wereimmediately transferred to a vial containing a fresh salt solutionconsisting of 126 mM NaCl, 3 mM KCl, 1.25 mM NaH₂PO₂ 26 mM NaHCO₃ 2 mMMgSO₄, 2 mM CaCl₂ and 10 mM D-glucose, aerated with 95% O₂ and 5% CO₂ atroom temperature. After 1 hour, electrophysiological recordings weremade. In the injured nerve, recordings were made in a segment distal tothe injury site. This segment contains axons of viable RGCs that haveescaped both primary and secondary damage, as well as the distal stumpsof non-viable RGCs that have not yet undergone Wallerian degeneration.The nerve ends were connected to two suction Ag—AgCl electrodes immersedin the bathing solution at 37° C. A stimulating pulse was appliedthrough the electrode, and the CAP was recorded by the distal electrode.A stimulator (SD9; Grass Medical Instruments, Quincy, Mass.) was usedfor supramaximal electrical stimulation at a rate of 1 pps to ensurestimulation of all propagating axons in the nerve. The measured signalwas transmitted to a microelectrode AC amplifier (model 1800; A-MSystems, Everett, Wash.). The data were processed using the LabView2.1.1 data acquisition and management system (National Instruments,Austin, Tex.). For each nerve, the difference between the peak amplitudeand the mean plateau of eight CAPs was computed and was considered asproportional to the number of propagating axons in the optic nerve. Theexperiments were done by experimenters “blinded”, to sample identity. Ineach experiment the data were normalized relative to the mean CAP of theuninjured nerves from PBS-injected rats.

[0176] Clinical Evaluation of Experimental Autoimmune Encephalomyelitis(EAE)

[0177] Clinical disease was scored every 1 to 2 days according to thefollowing neurological scale: 0, no abnormality; 1, tail atony; 2, hindlimb paralysis; 3, paralysis extending to thoracic spine; 4, front limbparalysis; 5, moribund state.

Results

[0178] Neuroprotection by Autoimmune Anti-MBP T Cells

[0179] Morphological analyses were done to assess the effect of the Tcells on the response of the nerve to injury, and specifically onsecondary degeneration. Rats were injected intraperitoneally immediatelyafter optic nerve injury with PBS or with 1×10⁷ activated T cells of thevarious cell lines. The degree of primary damage to the optic nerveaxons and their attached RGCs was measured by injecting the dye4-Di-10-Asp distal to the site of the lesion immediately after theinjury. A time lapse of 2 weeks between a moderate crush injury and dyeapplication is optimal for demonstrating the number of still viablelabeled neurons as a measure of secondary degeneration, and as theresponse of secondary degeneration to treatment. Therefore, secondarydegeneration was quantified by injecting the dye immediately or 2 weeksafter the primary injury, and calculating the additional loss of RGCsbetween the first and the second injections of the dye. The percentageof RGCs that had survived secondary degeneration was then calculated.The percentage of labeled RGCs (reflecting still-viable neurons) wassignificantly greater in the retinas of the rats injected with anti-MBPT cells than in the retinas of the PBS-injected control rats (FIG. 2).In contrast, the percentage of labeled 30 RGCs in the retinas of therats injected with anti-OVA or anti-p277 T cells was not significantlygreater than that in the control retinas. Thus, although the three Tcell lines accumulated at the site of injury, only the MBP-specificautoimmune T cells had a substantial effect in limiting the extend ofsecondary degeneration. Labeled RGCs of injured optic nerves of ratsinjected with PBS (FIG. 3A), with anti-p277 T cells (FIG. 3B) or withanti-MBP T cells (FIG. 3C) were compared morphologically usingmicrographs.

[0180] Clinical Severity of EAE

[0181] Animals were injected i.p. with 10⁷ T_(MBP) cells with or withoutconcurrent optic nerve crush injury. The clinical course of the ratsinjected with the T_(MBP) cells was evaluated according to theneurological paralysis scale. Each group contained 5-9 rats. Thefunctional autoimmunity of the injected anti-MBP T cells wasdemonstrated by the development of transient EAE in the recipients ofthese cells. As can be seen in FIG. 4A, the course and severity of theEAE was not affected by the presence of the optic nerve crush injury.

[0182] Survival of RGCS in Non-Injured Nerves

[0183] Animals were injected i.p. with 10⁷ T_(MBP) cells or PBS. Twoweeks later, 4-Di-10-Asp was applied to the optic nerves. After fivedays the retinas were excised and flat mounted. Labeled RGCs from fivefields (located at approximately the same distance from the optic disk),in each retina were counted and their average number per are (mm²) wascalculated.

[0184] As can be seen in FIG. 4B, there is no difference in the numberof surviving RGCs per area (mm²) in non-injured optic nerves of ratsinjected with anti-MBP T cells compared to in rats injected with PBS.

[0185] Neuroprotection by T Cells Reactive to a CrypticEpitope—(p51-70)MBP

[0186] To determine whether the neuroprotective effect of the anti-MBP Tcells is correlated with their virulence, the effect of T cells reactiveto a “cryptic” epitope of MBP, the peptide 51-70 (p51-70) was examined.“Cryptic” epitopes activate specific T cells after an animal isimmunized with the particular peptide, but not with the whole antigen(Mor et al, 1995). The T cell line reactive to the whole MBP and the Tcell line reactive to the cryptic epitope p51-70 were compared for theseverity of the EAE they induced, and for their effects on secondarydegeneration. In rats injected with the T cell line reactive to thecryptic epitope, disease severity (as manifested by the maximal EAEscore) was significantly lower than that in rats injected with the Tcell line reactive to the whole protein (Table 1). Whereas anti-MBP Tcells caused clinical paralysis of the limbs, rats injected with theanti-p51-70 T cells developed only tail atony, not hind limb paralysis,and almost none showed weakness of the hind limbs. Despite thisdifference in EAE severity, the neuroprotective effect of the lessvirulent (anti-p51-70) T cells was similar to that of the more virulent(anti-MBP) T cells (FIG. 5). The percentage of RGCs surviving secondarydegeneration in the retinas of rats injected with either of the lineswas significantly higher than in the retinas of the PBS-injected rats.Thus, there was no correlation between the neuroprotective effect of theautoimmune T cells and their virulence. It is possible that theanti-p51-70 T cells encounter little antigen in the intact CNS, andtherefore cause only mild EAE. Their target antigen may however becomemore available after injury, enabling these T cells to exert aneuroprotective effect. TABLE 1 Anti-MBP and Anti-p501-70 T Cells Varyin Pathogenicity T Cell Line Clinical EAE Mean Max. Score Whole MBPModerate to Severe 2.00 + 0.2 p51-70 of MBP Mild 0.70 + 0.2 # the meanmaximal score of rats injected with anti-MBP T cells and that of ratsinjected with anti-p51-70 T cells (P = 0.039, Student's t-test).

[0187] Electrophysiological Activity

[0188] To confirm the neuroprotective effect of the anti-MBP T cells,electrophysiological studies were done. Immediately after optic nerveinjury, the rats were injected intraperitoneally with PBS or with 1×10⁷activated anti-MBP or anti-OVA T cells. The optic nerves were excised 7,11 or 14 days later and the CAPs, a measure of nerve conduction, wererecorded from the injured nerves. On day 14, the mean CAP amplitudes ofthe distal segments recorded from the injured nerves obtained from thePBS-injected control rats were 33% to 50% of those recorded from therats injected with the anti-MBP T cells (FIG. 6A, Table 2). As thedistal segment of the injured nerve contains both neurons that escapedthe primary insult and injured neurons that have not yet degenerated,the observed neuroprotective effect could reflect the rescue of sparedneurons, or a delay of Wallerian degeneration of the injured neurons(which normally occurs in the distal stump), or both. No effect of theinjection of anti-MBP T cells on the mean CAP amplitudes of uninjurednerves was observed (FIG. 6B, Table 2). It is unlikely that theneuroprotective effect observed on day 14 could have been due to theregrowth of nerve fibers, as the time period was too short for this.

[0189] The strong neuroprotective effect of the anti-MBP T cells seen onday 14 was associated with a significantly decreased CAP amplituderecorded on day 7 (Table 2). The anti-MBP T cells manifested nosubstantial effect on the uninjured nerve on day 7, indicating that thereduction in electrophysiological activity observed in the injured nerveon day 7 might reflect the larger number of T cells present at theinjury site relative to the uninjured nerve (FIG. 1). The observedreduction in CAP amplitude in the injured nerve on day 7 reflected atransient resting state in the injured nerve. This transient effect hasnot only disappeared, but was even reversed by day 14 (Table 2). Earlysigns of the neuroprotective effect could already be detected on day 11in the rats injected with anti-OVA T cells, no reduction in CAPamplitude on day 7 could be detected in either the injured or theuninjured nerves, and no neuroprotective effect was observed on day 14(Table 2). Thus, it seems that the early reduction in CAP and the lateneuroprotection shown specifically by the anti-MBP T cells are related.TABLE 2 Transient Reduction in Electrophysiological Activity of theInjured Optic Nerve Induced by Anti-MBP T Cells, Followed by aNeuroprotective Effect Uninjured Optic Nerve Injured Optic Nerve Day 7Day 14 Day 7 Day 14 Ratio (%)  89.9 ± 9.4 101.2 ± 22.7 63.8* ± 14.9243.1** ± 70.8 T_(MPB)/PBS (n = 22) (n = 10) (n = 17) (n = 8) Ratio (%)109.7 ± 13.  92.5 ± 12.6 125.5 ± 24.4  107.3 ± 38.9 T_(OVA)/PBS (n = 11)(n = 3) (n = 11) (n = 4) # injected rats/mean CAP of injured nerves fromPBS-injected rats) × 100. The P value was calculated by comparing thelogarithms of the normalized CAP amplitudes of nerves from PBS-injectedrats and rats injected with T cells, using the unpaired Student's test,*P <0.05; **P <0.001 n = sample size.

[0190] Neuroprotection in Spinal Cord Injury by Anti-MBP T Cells

[0191] Materials and Methods

[0192] Animals, antigens (MBP, OVA) and T cell lines were as describedhereinbefore in Example 6 animals, antigens and T cell lines,respectively.

[0193] Contusion. Adult rats (300 to 350 g) were anesthetized and thespinal cord was exposed by laminectomy at the level of T7-T8. One hourafter induction of anesthesia, a 10-gram rod was dropped onto thelaminectomized cord from a height of 50 mm. The impactor device(designed by Prof. Wise Young) allowed, for each animal, measurement ofthe trajectory of the rod and its contact with the spinal cord to allowuniform lesion. Within an hour of the contusion, rats were injectedi.p., on a random basis, with either 10⁷ cells (specific to either MBPor OVA, depending on the experimental design) or with PBS. Bladderexpression was done at least twice a day (particularly during the first48 h after injury, when it was done 3 times a day) until the end of thesecond week, by which time the rats had developed autonomous bladdervoidance. Approximately twice a week, locomotor activity (of the trunk,tail and hind limbs) in an open field was evaluated by placing the ratfor 4 min in the middle of a circular enclosure made of molded plasticwith a smooth, non-slip floor (90 cm diameter, 7 cm wall height).

[0194] Results

[0195] The present study of spinal cord neuroprotection was prompted bythe previous example that partial injury to an optic nerve can beameliorated administering T cells directed to a CNS self-antigen. Thequestion was whether autoimmune T cells could have a beneficial effecton recovery from traumatic spinal cord injury with its greater mass ofinjured CNS tissue and the attendant spinal shock.

[0196] Adult Lewis rats were subjected to a calibrated spinal cordcontusion produced by dropping a 10-gram weight from a height of 50 mmonto the laminectomized cord at the level of T7-T8 (see Basso et al,1996). The rats were then injected intraperitoneally with autoimmune Tcells specific to MBP. Control rats were similarly injured but receivedeither no T cells or T cells specific to the non-self antigen ovalbumin(OVA). Recovery of the rats was assessed every 3 to 4 days in terms oftheir behavior in an open-field locomotion test, in which scores rangefrom 0 (complete paraplegia) to 21 (normal mobility). The locomotorperformance of the rats was judged by observers blinded to the identityof the treatment received by the rats. Included in the study was a groupof uninjured, sham-operated (laminectomized but not contused) rats thatwere injected with anti-MBP T cells to verify the activity of the Tcells. In all the sham-operated rats, the anti-MBP T cells inducedclinical EAE, which developed by day 4, reached a peak at day 7 andresolved spontaneously by day 11. Note, therefore, that at the earlypost-traumatic stage, any effect of the autoimmune T cells on theinjured spinal cord, whether positive or negative, would be transientlymasked both by spinal shock and by the paralysis of EAE.

[0197] Indeed, none of the rats with contused spinal cords showed anylocomotor activity in the first few days after the contusion (FIG.7A).Interestingly, however, the rats treated with anti-MBP T cells recoveredearlier from spinal shock; on day 11, for example, when no recoverycould be detected in any of the untreated control rats, significantimprovement was noted in the T cell-treated rats (FIG. 7A). At all timepoints thereafter, the rats that had received the autoimmune T cellsshowed better locomotor recovery than did the untreated injured rats(FIG. 7A). Thus the autoimmune T cells, in spite of beingencephalitogenic, did confer significant neuroprotection. Moreover, thephase of neuroprotective activity coincided with the phase of immuneparalysis, supporting our suggestion that neuroprotection might berelated to transient paralysis.

[0198] By one month after trauma the rats in both groups had reached amaximal behavioral score, which then remained at plateau for at least 3months of follow-up. In the untreated rats, maximal recovery oflocomotor behavior, as noted in previous reports of similarly severecontusion (Basso et al, 1996), was marked by some ineffectual movementof hind-limb joints, but the rats showed no ability to support theirbody weight and walk, and obtained a score of 7.3±0.8 (mean±SEM). Incontrast, the average score of the rats that had been treated with theanti-MBP T cells was 10.2±0.8, and in some rats the value was as high as13. All the rats in the treated group could support their body weightand some could frequently walk in a coordinated fashion. The differencebetween the two groups, based on 2-factor repeated ANOVA, wasstatistically significant (p<0.05). The recovery curve based onlocomotor activity is nonlinear. The above-described increase in motoractivity seen after treatment with the anti-MBP T cells could resultfrom much higher percentage of spared tissue based on a linearregression curve on which the behavioral score is correlated with theamount of neural spinal cord tissue (for example, a difference between11 and 7 on the locomotion score would be read as a difference between30% and less than 10% of spared tissue).

[0199] In another set of experiments the rats were subjected to a moresevere insult, resulting in a functional score of 1.9±0.8 (mean±SEM) inthe untreated group and 7.7±1.4 in the treated group (FIG. 7B). Thisdifference of more than 3 fold in behavioral scores was manifested bythe almost total lack of motor activity in the control rats as comparedwith the ability of the autoimmune T cell-treated rats to move all theirjoints. The beneficial effect was specific to treatment with anti-MBP Tcells; no effect was observed after treatment with T cells specific tothe non-self antigen OVA (data not shown). The positive effect of theautoimmune T cells seems to be expressed in the preservation of CNStissue that escaped the initial lesion, i.e., in neuroprotection.Therefore, the magnitude of the effect would be inherently limited bythe severity of the insult; the more severe the lesion, the less theamount of spared tissue amenable to neuroprotection.

[0200] To determine whether clinical recovery could be explained interms of preservation of spinal axons, we performed retrograde labelingof the descending spinal tracts by applying the dye rhodamine dextranamine (Brandt et al, 1992) at T12, below the site of damage. The numberof dye-stained cells that could be counted in the red nucleus of thebrain constituted a quantitative measure of the number of intact axonstraversing the area of contusion. Sections of red nuclei from injuredrats treated with anti-MBP T cells (FIG. 8) contained 5-fold morelabeled cells than sections taken from the untreated injured rats.Photomicrographs of red nuclei taken from rats treated with anti-MBP Tcells (with an open field score of 10) and from PBS-treated rats (with ascore of 6) are shown in FIG. 8. These findings indicate that thereduction in injury-induced functional deficit observed in the Tcell-treated rats can be attributed to the sparing of spinal tracts,resulting in a higher degree of neuron viability.

[0201] After a follow-up of more than 3 months, when the locomotoractivity scores had reached a plateau, the site of injury of three ofPBS-treated animals and three animals treated with anti-MBP T cells wereanalyzed by diffusion-weighted MRI. The cords were excised in one piecefrom top to bottom and were immediately placed in fixative (4%paraformaldehyde). Axial sections along the excised contused cord wereanalyzed. FIG. 9 shows the diffusion anisotropy in axial sections alongthe contused cord of a rat treated with autoimmune T cells, as comparedwith that of PBS-treated control rat. The images show anisotropy in thewhite matter surrounding the gray matter in the center of the cord.Sections taken from the lesion sites of PBS-treated control rats showlimited areas of anisotropy, which were significantly smaller than thoseseen at comparable sites in the cords of the rats treated with theanti-MBP T cells. Quantitative analysis of the anisotropy, reflectingthe number of spared fibers, is shown in FIG. 9. The imaging resultsshow unequivocally that, as a result of the treatment with theautoimmune anti-MBP T cells, some spinal cord tracts had escaped thedegeneration that would otherwise have occurred.

[0202] Discussion of Results

[0203] No cure has yet been found for spinal cord lesions, one of themost common yet devastating traumatic injuries in industrial societies.It has been known for more that 40 years that CNS neurons, unlikeneurons of the PNS, possess only a limited ability to regenerate afterinjury. During the last two decades, attempts to promote regenerationhave yielded approaches that lead to partial recovery. In the last fewyears it has become apparent that, although most of the traumaticinjuries sustained by the human spinal cord are partial, the resultingfunctional loss is nevertheless far worse than could be accounted for bythe severity of the initial insult; the self-propagating process ofsecondary degeneration appears to be decisive.

[0204] A substantial research effort has recently been directed toarresting injury-induced secondary degeneration. All attempts up to nowhave been pharmacologically based, and some have resulted in improvedrecovery from spinal shock. The present study, in contrast, describes acell therapy that augments what seems to be a natural mechanism ofself-maintenance and leads, after a single treatment, to long-lastingrecovery. The extent of this recovery appears to exceed that reportedusing pharmacological methods.

[0205] In most tissues, injury-induced damage triggers a cellular immuneresponse that acts to protect the tissue and preserve its homeostasis.This response has been attributed to macrophages and other cellscomprising the innate arm of the immune system. Lymphocytes, which areresponsible for adaptive immunity, have not been thought to participatein tissue maintenance. Adaptive immunity, according to traditionalteaching, is directed against foreign dangers. Our studies now show,however, that the adaptive T cell immune response can be protective evenwhen there is no invasion by foreign pathogens. In the case of tissuemaintenance, the specificity of the T cells is to tissue self-antigens.

[0206] Our observation of post-traumatic CNS maintenance by autoimmune Tcells suggests that we might do well to reevaluate some basic conceptsof autoimmunity. T cells that are specific to CNS self antigens ingeneral, and to MBP in particular, have long been considered to be onlydetrimental to health. In the present study, however, the same T cellpreparation that can produce EAE in the undamaged CNS was found to beneuroprotective in the damaged spinal cord, suggesting that the contextof the tissue plays an important part in determining the outcome of itsinteraction with T cells. It would seem that the tissue deploys specificsignals to elicit particular T cell behaviors. Among such signals areco-stimulatory molecules, particularly members of the B7 family (Lenchowet al, 1996). As shown hereinafter, the injured rat optic nervetransiently expresses elevated levels of the co-stimulatory moleculeB7.2, which is constitutively expressed at low levels in the rat CNSwhite matter and which is thought to be associated with regulation ofthe cytokine profile of the responding T cells (Weiner, 1997). The earlypost-injury availability of the exogenous anti-MBP T cells, coincidingwith the observed early post-injury increase in B7.2, would support theidea that signals expressed by the tissue might modulate the T cellresponse. It is thus conceivable that anti-MBP T cells which cause amonophasic autoimmune disease upon interacting with a healthy CNS nerve,might implement a maintenance program when they interact with damagedCNS tissue expressing increased amounts of B7.2 and probably otherco-stimulatory molecules. The neuroprotective effects of the T cells maybe mediated, at least in part, by antigen-dependent regulation ofspecific cytokines or neurotrophic factors (Kerschensteiner et al, 1999)produced locally at the site of injury.

[0207] Thus, the present invention is also directed to manipulating B7.2co-stimulatory molecule to prevent or inhibit neuronal degeneration andameliorate the effects of injury to or disease of the nervous system.B7.2 molecule can be up-regulated for this purpose, using drugs or bygenetic manipulation, without undue experimentation.

[0208] In a recent study, it was reported that injury to the spinal cordtriggers a transient autoimmune response to MBP (Popovich et al, 1996).However, whether that response is detrimental or beneficial remained anopen question (Popovich et al, 1997). From our present data, it wouldappear that the activation of anti-MBP T cells could indeed bebeneficial. However, a supplement of exogenous autoimmune T cells may berequired to overcome the restrictions on immune reactivity imposed bythe immune-privilege of the CNS (Streilein, 1995). The finding thatautoimmune response can be advantageous suggests that natural autoimmuneT cells may have undergone positive selection during ontogeny, asproposed by the theory of the immunological homunculus (Cohen, 1992),and are not merely a default resulting from the escape from negativeselection of T cells that recognize self antigens (Janeway, 1992). Sucha response could then be considered as a mechanism of potentialphysiological CNS self-maintenance, which is, however, not sufficientfor the purpose because of the immune-privileged character of the CNS.

[0209] A single injection of autoimmune T cells lasted for at least 100days. Thus, this procedure offers a form of self-maintenance. Thisspecific autoimmune response, when properly controlled, is useful aspart of a self-derived remedy for spinal cord injury.

EXAMPLE 3

[0210] Neuroprotective Effects of a NS-Specific Antigen Peptide—MOGp35-55

Materials and Methods

[0211] Animals, crush injury of rat optic nerve, and retrograde labelingare described above in Examples 3 and 4. A peptide based on amino acids35-55 of MOG (MOG p35-55) was chemically synthesized at the WeizmannInstitute, Israel.

[0212] Inhibition of Secondary Degeneration

[0213] Rats were injected intradermally in the footpads with MOG p35-55(50 μg/animal) and IFA, or PBS, ten days prior to optic nerve crushinjury. RGCs were assessed two weeks after injury using retrogradelabeling as described above. The number of RGCs in rats injected withPBS or MOG p35-55 was expressed as a percentage of the total number ofneurons in rats injected with MOG p35-55 in the absence of crush injury.

[0214] Results

[0215] As shown in FIG. 10, the number of labeled RGCs (indicatingviable axons) was about 12.5 fold greater in animals injected with MOGp35-55 compared to animals receiving PBS.

EXAMPLE 4

[0216] Neuroprotective Effects of MBP Administered Orally

Materials and Methods

[0217] Animals, crush injury of rat optic nerve, and retrograde labelingof RGCs are described above in Examples 3 and 4.

[0218] Inhibition of Secondary Degeneration

[0219] Bovine MBP (Sigma, Israel) (1 mg/dose) was administered to ratsby gavage using a blunt needle. MBP was administered 5 times, everythird day, beginning 2 weeks prior to optic nerve crush injury. Thenumber of RGCs in treated animals was expressed as a percentage of thetotal number of neurons in animals subjected to optic nerve crush injurybut which did not receive MBP.

Results

[0220] As shown in FIG. 11, the number of labeled RGCs was about 1.3fold greater in animals treated with MBP compared to untreated animals.

[0221] The B7.2 Co-Stimulatory Molecule is Associated withPost-Traumatic Maintenance of the Optic Nerve by Oral Administration ofMBP

[0222] Introduction

[0223] Autoimmune T cells can under certain conditions be beneficial totraumatized CNS axons. The effect of such T cells on the damaged tissuemight be influenced by the nature and amount of the co-stimulatorymolecules it expresses. We show that the B7.2 co-stimulatory molecule isconstitutively expressed in the intact rat optic nerve, and after injuryis up-regulated at the margins of the injury site. Pre-injury inductionof oral tolerance to MBP resulted in a further post-injury increase inB7.2 at the margins and at the injury site itself, as well as a betterpreservation of the traumatized nerve. Thus, B7.2 expression in thebrain and its up-regulated after trauma seem to be directly related topost-traumatic maintenance displayed by autoimmune T cells.

[0224] Neuronal injury in the CNS causes degeneration of directlydamaged fibers as well as of fibers that escaped the primary insult. Italso triggers a systemic response of autoimmune T cells to MBP thatmight affect the course of degeneration of the injured nerve. Whetherthe effect of these T cells on the nerve is detrimental or beneficialmay depend, in part, on the nature and level of the co-stimulatorymolecules expressed by the damaged tissue.

[0225] Several co-stimulatory molecules have recently been identified,including the B7 and CD40 molecules (Caux et al, 1994; Lenschow et al,1996). CD40 appears to be dominant during cell differentiation in thelymph nodes and B7 during activation of T cells in the target organ(Grewal et al, 1996).

[0226] The B7 co-stimulatory molecule is a member of the immunoglobulinsuperfamily that interacts with CD28 and CTLA-4 on T_(H) cells. Thereare two related forms of B7 (B7.1 and B7.2). Both molecules have asimilar organization of extracellular domains but markedly differentcytosolic domains. Both B7 molecules are expressed on antigen-presentingcells (APCs) such as dendritic cells, activated macrophages andactivated B cells as B7.1 or B7.2., which might preferentially supportactivation of the Th1 or the Th2 type of immune response, respectively(Kuchroo et al, 1995; Karandikar et al, 1998). We were thereforeinterested in determining the identity of B7 molecule subtype expressedin intact and injured CNS white matter, and its possible influence onthe course of the response to the injury.

Results

[0227] The co-stimulatory molecule expressed constitutively in theintact optic nerves of adult Lewis rats was identified as B7.2. (FIGS.12A, 12B). To examine the effects of neurotrauma on the expression of B7co-stimulatory molecules, we inflicted a mild crush injury on the opticnerves of Lewis rats and assessed the neural expression of B7 byimmunohistochemical analysis. The most striking effect of the injury wasseen on B7.2 expression manifested on post-injury day 3 by its elevationat the margins of the injury site (FIGS. 12C, D, E). In contrast,expression of B7.1 was not detected in the optic nerve either before or3 days after injury. On day 7, however, B7.1 was detectable at the siteof injury, having pattern reminiscent of that seen for macrophages ormicroglia (FIG. 12F).

[0228] Next, we attempted to determine whether the degenerative responseto optic nerve injury could be modified by peripheral manipulation ofthe immune system. The manipulation chosen was induction of oraltolerance, known to cause a “bystander” T cell immunosuppressive effect(Weiner et al, 1997b). Ingestion of low doses of MBP results in theactivation of T cells which, based on antigen recognition, secrete TGFas the dominant cytokine and thus favor an immune response of Th2/3 type(Chen et al, 1994).

[0229] Lewis rats were fed with food to which 1 mg of bovine MBP hadbeen added five times daily every other day. Ten days after firstreceiving the supplement, the rats were subjected to mild unilateraloptic nerve crush injury. This time, interval between initiation of oraltolerance and injury was chosen to allow adequate build-up of thesystemic T cell response. As shown in FIGS. 13A and 13B, the numbers ofmacrophages or active microglia (indicated by ED-1 labeling) and T cells(indicated by immunolabeling for T cell receptor), assessed 3 days afterinjury, did not differ from those observed in control injured rats whichdid receive any treatment or were fed with PBS. In the rats with inducedoral tolerance to MBP, however, the amounts of B7.2 were furtherincreased at the margins of the site of injury (FIG. 13C) as comparedwith controls (FIG. 12E). In addition, in the rats with induced oraltolerance to MBP, B7.2 was also elevated at the site of injury relativeto the control nerves (FIG. 13C). It seems reasonable to assume that theT cells exposed to MBP via intestinal absorption, upon invading theinjured CNS, contributed to the increase in expression of B7.2 by theinjured nerve.

[0230] We then attempted to determine whether the observed changes inB7.2 expression in the injured rats was correlated with the extent ofneuronal degeneration. Acute injury of the rat optic nerve is followedby a process of nerve degeneration, which can be quantified byretrograde labeling of the surviving neurons and counting of thecorresponding cell bodies. Two weeks after optic nerve injury, thenumber of surviving RGCs, representing still-viable neurons, in thegroup of MBP-fed rats, was significantly higher than that in the controlgroup, or that in the group of rats with injured nerves that were fedwith ovalbumin (OVA). Interestingly, the benefit of the induced oraltolerance to MBP was increased by feeding the rats with more intensiveschedule (FIG. 14).

EXAMPLE 5

[0231] Posttraumatic Immunization with Nogo P472 Peptide PromotedFunctional Recovery from Spinal Cord Contusion

Material and Methods

[0232] Animals. Female and male SPD rats were supplied by the AnimalBreeding Center of the Weizmann Institute of Science, Rehovot, Israel,matched for age (8-12 weeks) and housed in light- andtemperature-controlled rooms.

[0233] Antigen. The Nogo p472 peptide (SEQ ID NO:19) was synthesized atthe Weizmann Institute of Science, Rehovot, Israel.

[0234] Immunization. Rats were immunized with 100-150 μg of Nogo p472peptide emulsified in CFA containing 1 mg/ml Mycobacterium tuberculosis.The emulsion was injected subcutaneously at one site in the upper backin the rats. The p472-immunized rats were boosted one week after injurywith p472 (100 μg/rat) in IFA. Control rats were injected either withPBS or with PBS emulsified in CFA (Difco, Detroit, Mich., USA).

[0235] Contusion. Adult rats were anesthetized and their spinal cordswere exposed by laminectomy at the level of T9. One hour after inductionof anesthesia, a 10-g rod was dropped onto the laminectomized cord froma height of 25 mm or 50 mm, using the NYU impactor (Basso et al, 1995and 1996).

[0236] Introduction

[0237] Regeneration of axons after injury in the CNS of highervertebrates is extremely limited and almost absent. Growth inhibitorsassociated with CNS myelin play an important role in this aspect. Apotent neurite growth inhibitory activity associated with adult CNSoligodendrocytes and myelin was reported by Caroni and Schwab, 1988, andfound to be neutralized by a monoclonal antibody, IN-1, which was shownto promote axonal regeneration and to enhance compensatory plasticityfollowing spinal cord or brain lesions in adult rats.

[0238] This activity was later related to a high molecular weightmembrane protein, designated NI-250, with a smaller component, NI-35, inrat. The bovine homologue of rat NI-250, bNI-220, was recently purified(Chen et al, 2000; PCT Publication WO 00/31235). The cloning of nogo A,the rat cDNA encoding NI-220/250, was recently reported (see FIG. 1a ofChen et al, 2000; and PCT Publication WO 00/31235, the entire contentsof both of which being hereby incorporated herein by reference). The ratnogo gene (SEQ ID NO: 17) encodes at least three major protein products:Nogo-A (SEQ ID NO:18) (1,163 amino acids; database accession numberAJ242961), Nogo-B (SEQ ID NO:20) (360 amino acids; AJ242962) and Nogo-C(SEQ ID NO:21) (199 amino acids; AJ242963). The sequence of the aminoacid p472 (SEQ ID NO:19) containing the residues 623-640 of rat Nogo-A,is shown in the box in FIG. 1a of Chen et al, 2000. The cloning of thecorresponding human cDNA and protein is reported in Prinjha et al, 2000.See also WO 00/60083 and WO 01/36631.

[0239] PCT Publication WO 00/31235 describes methods for the productionof recombinant Nogo proteins, fragments, derivatives and analogsthereof, and DNA molecules coding therefor. This publication furtherdescribes the use of a Nogo protein or fragment thereof for thetreatment of neoplastic diseases of the CNS such as glioma,glioblastoma, retinoblastoma, and the like; and further describes theuse of a ribozyme or an antisense Nogo nucleic acid for treatment of asubject with damage to the CNS and/or for inducing regeneration ofneurons, wherein said ribozyme or antisense Nogo nucleic acid acts byinhibiting the production of Nogo in the subject. It is thus unexpectedthat immunization with Nogo or fragments thereof and/or administrationof T cells activated therewith can promote nerve regeneration or preventor inhibit neuronal degeneration in the NS, as shown according to thepresent invention.

[0240] PCT publication WO 00/31235, WO 00/60083 and WO 01/36631 are allhereby incorporated herein by reference. All CNS protein polypeptidesand nucleotides disclosed herein can be used in the process of thepresent invention.

Results

[0241] SPD male rats (n=5 per group) were subjected to severe spinalcord contusion as described in the Materials and Methods section of thisexample and a 10-g rod was dropped onto the laminectomized cord from aheight of 50 mm (FIG. 24A) or 25 mm (FIG. 24B) using the NYU impactor.Soon thereafter the rats were immunized subcutaneoulsy with Nogo p472peptide (100 μg/rat) emulsified in CFA containing 1 mg/ml Mycobacteriumtuberculosis. Control male rats (n=5 per group) were injected with PBSemulsified in CFA.

[0242] In another experiment, 5 female rats were subjected to severespinal cord contusion as described in the Materials and Methods sectionof this example and a 10-g rod was dropped onto the laminectomized cordfrom a height of 50 mm (FIG. 25) using the NYU impactor. Soon thereafterthe rats were immunized subcutaneoulsy with Nogo p472 peptide (100μg/rat) emulsified in CFA containing 1 mg/ml Mycobacterium tuberculosis.Control female rats (n=5 per group) were injected with PBS emulsified inCFA or with PBS alone. P472-immunized rats were boosted one week afterinjury and immunization with p472 (100 μg/rat) in IFA.

[0243] Acute incomplete spinal cord injury at the low thoracic levelscauses an immediate loss of hindlimb motor activity that spontaneouslyrecovers within the first 12 days post-injury and stabilizes ondeficient movement abilities. The amount of motor function restorationis the sum up effect of the positive recovery from spinal shock and thenegative effect of longitudinal and ventral spread of damage. Atherapeutic approach aiming at reducing the spread of damage throughneuroprotection will result in a better recovery in terms of hindlimbmotor activity. The hind limb motor skills of the animals were scoredusing the BBB scoring method developed by Basso et al, 1996, followingthe kinetics and amount of hindlimb motor activity in the twoexperimental groups.

[0244] The results of the experiments above, depicted in FIGS. 24(A-B)and 25, show that both female (squares, FIG. 25) and male (triangles,FIGS. 24A-B) p472-immunized rats showed significantly improved overallfunctional recovery compared to the control rats injected with PBS inCFA (squares, FIGS. 24A-B and circles, FIG. 25) or PBS only (triangles,FIG. 25) (P<0.01, one way repeated measurements ANOVA). Males (FIGS.24A-B) showed significantly better locomotor performance thanPBS+CFA-treated controls from day 11 after the injury and at all timespoints measured thereafter. Females (FIG. 25) showed significantlybetter hind limb locomotion than PBS-treated controls from day 25 and on((P≦0.05, **P≦0.01, ***P≦0.001, two-tail Student T-test).

[0245] Discussion of Experimental Results

[0246] The results of the experiments described in Examples 1 and 2 showthat activated T cells accumulate at a site of injury in the CNS.Furthermore, the results also demonstrate that the accumulation of Tcells at the site of injury is a non-specific process, i.e., T cellswhich accumulated at the site of injury included both T cells which areactivated by exposure to an antigen present at the site of injury aswell as T cells which are activated by an antigen not normally presentin the individual.

[0247] The results of experiments described in Example 3 demonstratethat the beneficial effects of T cells in ameliorating damage due toinjury in the CNS are associated with an NS-specific self-antigen asillustrated by MBP. More specifically, the administration ofnon-recombinant T cells which were activated by exposure to an antigenwhich can cause autoimmune disease (T_(MBP)), rather than aggravatingthe injury, led to a significant degree of protection from secondarydegeneration. Thus, activating T cells by exposure to a fragment of anNS-specific antigen was beneficial in limiting the spread of injury inthe CNS. The present findings show that secondary degeneration can beinhibited by the transfer into the individual on non-recombinant T cellswhich recognize an NS-specific self antigen which is present at a siteof injury. The T cells may recognize cryptic or non-pathogenic epitopesof NS-self antigens.

[0248] In addition, the experiments described in Examples 3, 4 and 5show that activation of T cells by administering an immunogenic antigen(e.g., MBP) or immunogenic epitope of an antigen (e.g., MOG p35-55 orNogo p472), may be used for preventing or inhibiting secondary CNSdegeneration following injury.

[0249] The foregoing description of the specific embodiments will sofully reveal the general nature of the invention that others can, byapplying current knowledge, readily modify and/or adapt for variousapplications such specific embodiments without undue experimentation andwithout departing from the generic concept, and, therefore, suchadaptations and modifications should and are intended to be comprehendedwithin the meaning and range of equivalents of the disclosedembodiments. It is to be understood that the phraseology or terminologyemployed herein is for the purpose of description and not of limitation,and that other means or steps for carrying out the same function can beused; and it is intended that such expressions be given their broadestinterpretation.

[0250] All publications cited herein are incorporated by reference intheir entirety.

REFERENCES

[0251] Altschul, et al, 1990, J. Mol. Biol. 215(3):403-410.

[0252] Arquint et al, Proc. Natl. Acad. Sci. USA 84:600-604, 1987.

[0253] Ashwood-Smith, 1961, Nature 190:1204-1205.

[0254] Basso, D M, Beattie, M S and Bresnahan, J C, 1995, “A sensitiveand reliable locomotor rating scale for open field testing in rats, J.Neurotrauma 12(1):1-21.

[0255] Basso D M, Beattie, M S and Bresnahan, J C, 1996, “Gradedhistological and locomotor outcomes after spinal cord contusion usingthe NYU weight-drop device versus transection, Exp. Neurol. 139(2):244-256.

[0256] Bazan, N. G., Rodriguez de Turco, E. B., Allan, G., 1995,“Mediators of injury in neurotrauma: intracellular signal transductionand gene expression”, J. Neurotrauma 12:791-814.

[0257] Bender et al, 1960, J. Appl. Physiol. 15:520.

[0258] Ben-Nun, A., et al, 1981a, “The rapid isolation of clonableantigen-specific T-lymphocyte lines capable of mediating autoimmuneencephalomyelitis Eur. J. Immunol. 11:195-199.

[0259] Ben-Nun, A., Wekerle, H. and Cohen, I. R., 1981, “Vaccinationagainst autoimmune encephalomyelitis with T-lymphocyte line cellsreactive against myelin basic protein”, Nature 292:60-61.

[0260] Ben-Nun, A. and Cohen, I. R., 1982, “Experimental autoimmuneencephalomyelitis (EAE) mediated by T-cell lines: process of selectionof lines and characterization of the cells”, J. Immunol. 129:303-308.

[0261] Bone-Marrow Conservation, Culture and Transplantation,Proceedings of a Panel, Moscow, Jul. 22-26, 1968, International AtomicEnergy Agency, Vienna, pp. 107-186.

[0262] Brandt et al, 1992, J. Neurosci. Methods 45:35-40.

[0263] Burns, J., Rosenzweig, A., Zweiman, B., Lisak, R. P., 1983,Isolation of myelin basic protein-reactive T-cell lines from normalhuman blood”, Cell Immunol. 81:435-440.

[0264] Caroni, P. and Schwab, M. E., 1988, “Two membrane proteinfractions from rat central myelin with inhibitory properties for neuritegrowth and fibroblast spreading” J. Cell Biol. 106: 1281-1288.

[0265] Caux et al, 1994, “Activation of Human Dendritic Cells throughCD40 Cross-Linking”, J. Exp. Med. 180:1263-1272.

[0266] Chen, Y., Kuchroo, V. K.,. Inobe, J. Hafler, D. A. & Weiner, H.L., 1994, “Regulatory T cell clones induced by oral tolerance:suppression of autoimmune encephalomyelitis” Science 265:1237-1240.

[0267] Chen, M. S., Huber, A. B., van der Haar, M. E., Schwab, M. E.,2000, “Nogo-A is a myelin-associated neurite outgrowth inhibitor and anantigen for monoclonal antibody IN-1”, Nature 403:434-439.

[0268] Cohen, I. R., 1992, Immunol. Today 13, 490-494.

[0269] Diehl et al, Proc. Natl. Acad Sci. U.S.A. 83(24):9807-9811, 1986(published erratum appears in Proc Natl Acad Sci U.S.A. 86(6):617-8,1991).

[0270] Duvdevani et al, 1990, Neurol. Neurosci. 2:31-38.

[0271] Elias et al, 1991, Proc. Natl. Acad. Sci. USA 88:3088-3091.

[0272] Faden, A. I., et al, 1992, Trends Pharmacol. Sci. 13:29-35.

[0273] Faden, A. I., 1993, “Experimental neurobiology of central nervoussystem trauma”, Crit. Rev. Neurobiol. 7:175-186.

[0274] Fournier, A. E., GrandPre, T., Strittmatter, S. M., 2001,“Identification of a receptor mediating Nogo-66 inhibition of axonalregeneration”, Nature 409:341.

[0275] Gonzalez et al, Mol. Phylogent. Evol. 6:63-71, 1996.

[0276] Gorin, Clinics in Haematology, 1986, 15(1):19-48.

[0277] Grewal et al, 1996, “Requirement for CD40 Ligand inCo-stimulation Induction, T Cell Activation, and Experimental AllergicEncephalomyelitis”, Science 273:1864-1867.

[0278] Hauben, E. et al, 2000, “Passive or active immunization withmyelin basic protein promotes recovery from spinal cord contusion”, J.Neurosci. 20:6421-6430.

[0279] Hickey, W. F. et al, 1991, “T-lymphocyte entry into the centralnervous system”, J. Neurosci. Res. 28:254-260.

[0280] Higgins, et al, 1996, Methods Enzymol 266:383-402.

[0281] Hirschberg, D. L., et al, 1998, “Accumulation of passivelytransferred primed T cells independently of their antigen specificityfollowing central nervous system trauma”, J. Neuroimmunol. 89:88-96.

[0282] Hirshfeld, et al, 1970, FEBS Lett. 7:317.

[0283] Hovda, D. A. et al, 1991, Brain Res. 567:1-10.

[0284] Hunig et al, 1989, “A monoclonal antibody to a constantdeterminant of the rat T cell antigen receptor that induces T cellactivation. Differential reactivity with subsets of immature and matureT lymphocytes”, J. Exp. Med., 169:73-86.

[0285] Hutchinson, C., et al, 1978, J. Biol. Chem 253:6551.

[0286] Janeway, C. A. Jr., 1992, “The immune system evolved todiscriminate infectious nonself from noninfectious self”, Immunol. Today13:11-16.

[0287] Karnholz et al, 1986, Proc. Natl. Acad. Sci. U.S.A.83(13):4962-4966.

[0288] Karandikar et al, 1998, “Targeting the B7/CD28:CTLA-4co-stimulatory system in CNS autoimmune disease”, J. Neuroimmunol.89:10-18.

[0289] Kerschensteiner, M. et al, 1999, J. Exp. Med. 189:865-870.

[0290] Kramer, R. et al, 1995, Nature Med. 1(11):1162-1166.

[0291] Kuchroo et al, 1995, “B7-1 and B7-2 co-stimulatory moleculesactivate differentially the Th1/Th2 developmental pathways: applicationto autoimmune disease therapy”, Cell 80:707-718.

[0292] Lazarov Spiegler, O., et al, 1996, FASEB J. 19:1296-1302.

[0293] Lenschow et al, 1996, “CD28/B7 System of T Cell co-stimulation”,Annu. Rev. Immunol. 14:233-258.

[0294] Lewis et al, 1967, Transfusion 7(1):17-32.

[0295] Linner et al, J. Histochem. Cytochem., 1986, 34(9):1123-1135.

[0296] Livesey and Linner, Nature, 1987, 327:255.

[0297] Lovelock, Biochem. J. 56:265, 1954.

[0298] Lovelock and Bishop, Nature 183:1394-1395, 1959.

[0299] Lynch, D. R. et al, 1994, “Secondary mechanisms in neuronaltrauma”, Curr. Opin. Neurol. 7:510-516.

[0300] Maniatis, T., 1990, Molecular Cloning, A Laboratory Manual, 2ded., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

[0301] Martin, R. et al, 1990, J. Immunol. 145:540-548.

[0302] Martin, R. 1997, J. Neural Transm. Suppl. 49:53-67.

[0303] Mazur, Science, 1970, 168:939-949.

[0304] McIntosh, T. K., 1993, :Novel pharmacologic therapies in thetreatment of experimental traumatic brain injury: a review”, J.Neurotrauma 10:215-261.

[0305] Moalem, G. et al, 1999, “Autoimmune T cells protect neurons fromsecondary degeneration after central nervous system axotomy”, NatureMed. 5: 49-55.

[0306] Mor et al, 1990, “Clinical modeling of T cell vaccination againstautoimmune diseases in rats. Selection of antigen-specific T cells usinga mitogen”, Clin. Invest. 85:1594-1598.

[0307] Mor et al, 1995, “Pathogenicity of T cells responsive to diversecryptic epitopes of myelin basic protein in the Lewis rat”, J. Immunol.155:3693-3699.

[0308] Nave et al, Proc. Natl. Acad. Sci. U.S.A 84:600-604, 1987.

[0309] Ota, K. et al, 1990, “T-cell recognition of an immunodominantmyelin basic protein epitope in multiple sclerosis”, Nature 346:183-187.

[0310] Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8.

[0311] Pette, M. et al, 1990, “Myelin basic protein-specific Tlymphocytes lines from MS patients and healthy individuals”, Proc. Natl.Acad. Sci. USA 87:7968-7972.

[0312] Phan The Tran and Bender, 1960, Proc. Soc. Exp. Biol. Med.104:388.

[0313] Phan The Tran and Bender, Exp. Cell Res. 20:651, 1960.

[0314] Phan The Tran and Bender, 1961, in Radiobiology, Proceedings ofthe Third Australian Conference on Radiobiology, Ilbery, P. L. T., ed.,Butterworth, London, p. 59.

[0315] Popovich, P. G., Stokes, B. T., Whitacre, D. C., 1996, “Conceptof autoimmunity following spinal cord injury: possible roles for Tlymphocytes in the traumatized central nervous system”, J. Neurosci.Res. 45:349-63.

[0316] Popovich et al, 1997, J. Comp. Neurol. 377:443-464.

[0317] Prinja et al, “Inhibitor of neurite outgrowth in humans”, Nature403(6768):383-384 (2000)

[0318] Rapalino, O., Lazarov-Spiegler, O., Agranov, E., Schwartz, M.,1998, “Implantation of stimulated homologous macrophages results inpartial recovery of paraplegic rats”, Nature Med. 4:814-821.

[0319] Rapatz et al, Cryobiology, 1968, 5(1):18-25.

[0320] Rinfret, Ann. N.Y. Acad. Sci. 85:576, 1960.

[0321] Roth et al, Genomics 28(2):241-250, 1995.

[0322] Rowe, Cryobiology, 1966, 3(1):12-18.

[0323] Rowe and Rinfret, 1962, Blood, 20:636.

[0324] Rowe et al, Fed. Proc., 1962, 21:157.

[0325] Schaich et al, 1986, Biol. Chem. 367:825-834.

[0326] Schluesener, H. J. and Wekerle, H., 1985, “Autoaggressive Tlymphocyte lines recognizing the encephatoligenic region of myelin basicprotein: in vitro selection from unprimed rat T lymphocyte populations”,J. Immunol. 135:3128-3133.

[0327] Sloviter and Ravdin, Nature 196:548, 1962.

[0328] Spitzer et al, Cancer, 1980, 45:3075-3085.

[0329] Stiff et al, Cryobiology, 1983, 20:17-24.

[0330] Streilein, J. W., 1993, Curr. Opin. Immunol. 5:428-423.

[0331] Streilein, J. W., 1995, Science 270:1158-1159.

[0332] Suruhan-Dires Keneli et al, 1993 Euro. J. Immunol. 23:530.

[0333] Thompson, et al, 1994, Nucleic Acids Res. 22(22):4673-80.

[0334] Vergelli, M. et al, 1996, “Differential activation of humanautoreactive T cell clones by altered peptide ligands derived frommyelin basic protein peptide (87-99)”, Eur. J. Immunol. 26: 2624-2634.

[0335] Weiner et al, 1997a, Annu. Rev. Med. 48:341-51.

[0336] Weiner et al, 1997b, “Tolerance Immune Mechanisms and Treatmentof Autoimmune Diseases”, Immunol. Today 18:335-343. Werkele, H., 1993,In The Blood-Brain Barrier, Pardridge, Ed., Raven Press, Ltd. New York,67-85.

[0337] Wu, D. et al, 1994, J.Neurochem. 62:37-44.

[0338] Yoles, E. et al, 1992, Invest. Ophthalmol. Vis. Sci.33:3586-3591).

[0339] Yoles et al, 1996, J. Neurotrauma 13:49-57.

[0340] Yoshina, A. et al, 1991 Brain Res. 561:106-119.

[0341] Zaroulis and Leiderman, Cryobiology 17:311-317, 1980.

[0342] Zivin, J. A., et al, 1991 Sci. Am. 265:56-63.

1 29 1 612 DNA Homo sapiens 1 ccaagaagat cccacagcag cttccgaaggcctggatgtg atggcatcac agaagagacc 60 ctcacagcga cacggatcca agtacttggccacagcaagt accatggacc atgcccggca 120 tggcttcctc ccaaggcaca gagacacgggcatccttgac tccatcgggc gcttctttag 180 cggtgacagg ggtgcgccca agcggggctctggcaaggac tcacacacaa gaactaccca 240 ctacggctcc ctgccccaga agtcgcagaggacccaagat gaaaacccag tagtccactt 300 cttcaagaac attgtgacac ctcgtacaccccctccatcc caaggaaagg ggagaggcct 360 gtccctcagc agatttagct ggggaggaagagacagccgc tctggatctc ccatggcaag 420 acgctgagag cctccctgct cagccttcccgaatcctgcc ctcggcttct taatataact 480 gccttaaacg tttaattcta cttgcaccaaatagctagtt agagcagacc ctctcttaat 540 cccgtggggc tgtgaacgcg gcgggccagcccacggcacc ctgactggct aaaactgttt 600 gtcccttttt at 612 2 2139 DNA Homosapiens 2 gaaaacagtg cagccacctc cgagagcctg gatgtgatgg cgtcacagaagagaccctcc 60 cagaggcacg gatccaagta cctggccaca gcaagtacca tggaccatgccaggcatggc 120 ttcctcccaa ggcacagaga cacgggcatc cttgactcca tcgggcgcttctttggcggt 180 gacaggggtg cgccaaagcg gggctctggc aaggactcac accacccggcaagaactgct 240 cactatggct ccctgcccca gaagtcacac ggccggaccc aagatgaaaaccccgtagtc 300 cacttcttca agaacattgt gacgcctcgc acaccacccc cgtcgcagggaaaggggaga 360 ggactgtccc tgagcagatt tagctggggg gccgaaggcc agagaccaggatttggctac 420 ggaggcagag cgtccgacta taaatcggct cacaagggat tcaagggagtcgatgcccag 480 ggcacgcttt ccaaaatttt taagctggga ggaagagata gtcgctctggatcacccatg 540 gctagacgct gaaaacccac ctggttccgg aatcctgtcc tcagcttcttaatataactg 600 ccttaaaact ttaatcccac ttgcccctgt tacctaatta gagcagatgacccctcccct 660 aatgcctgcg gagttgtgca cgtagtaggg tcaggccacg gcagcctaccggcaatttcc 720 ggccaacagt taaatgagaa catgaaaaca gaaaacggtt aaaactgtccctttctgtgt 780 gaagatcacg ttccttcccc cgcaatgtgc ccccagacgc acgtgggtcttcagggggcc 840 aggtgcacag acgtccctcc acgttcaccc ctccaccctt ggactttcttttcgccgtgg 900 ctcggcaccc ttgcgctttt gctggtcact gccatggagg cacacagctgcagagacaga 960 gaggacgtgg gcggcagaga ggactgttga catccaagct tcctttgtttttttttcctg 1020 tccttctctc acctcctaaa gtagacttca tttttcctaa caggattagacagtcaagga 1080 gtggcttact acatgtggga gctttttggt atgtgacatg cgggctgggcagctgttaga 1140 gtccaacgtg gggcagcaca gagagggggc cacctcccca ggccgtggctgcccacacac 1200 cccaattagc tgaattcgcg tgtggcagag ggaggaaaag gaggcaaacgtgggctgggc 1260 aatggcctca cataggaaac agggtcttcc tggagatttg gtgatggagatgtcaagcag 1320 gtggcctctg gacgtcaccg ttgccctgca tggtggcccc agagcagcctctatgaacaa 1380 cctcgtttcc aaaccacagc ccacagccgg agagtccagg aagacttgcgcactcagagc 1440 agaagggtag gagtcctcta gacagcctcg cagccgcgcc agtcgcccatagacactggc 1500 tgtgaccggg cgtgctggca gcggcagtgc acagtggcca gcactaaccctccctgagaa 1560 gataaccggc tcattcactt cctcccagaa gacgcgtggt agcgagtaggcacaggcgtg 1620 cacctgctcc cgaattactc accgagacac acgggctgag cagacggcccctgtgatgga 1680 gacaaagagc tcttctgacc atatccttct taacacccgc tggcatctcctttcgcgcct 1740 ccctccctaa cctactgacc caccttttga ttttagcgca cctgtgattgataggccttc 1800 caaagagtcc cacgctggca tcaccctccc cgaggacgga gatgaggagtagtcagcgtg 1860 atgccaaaac gcgtcttctt aatccaattc taattctgaa tgtttcgtgtgggcttaata 1920 ccatgtctat taatatatag cctcgatgat gagagagtta caaagaacaaaactccagac 1980 acaaacctcc aaatttttca gcagaagcac tctgcgtcgc tgagctgaggtcggctctgc 2040 gatccatacg tggccgcacc cacacagcac gtgctgtgac gatggctgaacggaaagtgt 2100 acactgttcc tgaatattga aataaaacaa taaactttt 2139 3 581DNA Homo sapiens 3 taatatctag ggktttgact ctgacccgtg ttggggctctcacttcatgg cttctcacgc 60 ttgtgctgca tatcccacac caattagacc caaggatcagttggaagttt ccaggacatc 120 ttcattttat ttccaccctc aatccacatt tccagatgtctctgcagcaa agcgaaattc 180 caggcaagcc ttagggaaaa aaggaaaaac aaagaaaatgaaacaattgg cagtgaaagg 240 cagaaagaga agatggagcc cttagagaag ggagtatccctgagtaggtg gggaaaaggg 300 gaggagaagg ggaggaggag aggaggagga aagcaggcctgtccctttaa gggggttggc 360 tgtcaatcag aaagcccttt tcattgcagg agaagaggacaaagatactc agagagaaaa 420 agtaaaagac cgaagaagga ggctggagag accaggatccttccagctga acaaagtcag 480 ccacaaagca gactagccag ccggctacaa ttggagtcagagtcccaaag acatgggtaa 540 gtttcaaaaa ctttagcatt gaagattcaa gaggacacag g581 4 1762 DNA Homo sapiens 4 ctgctttcag agcctgtgac ttcttgtgtgcctctcctgt ttctcagcaa catggcatag 60 ggcctgggat accaggtctg gggatctcagggactcttag cactttaaga cacatgtgtt 120 cccaggccct ggtgtgttcc tctagtgccagaaagatgtt tcatgctttg ctgactttgt 180 ataaagtctg tttgtagctg ttttgacagaatctcagcgt ataactgagg gtggggacat 240 tagccaagct gcattatagg aggacaaaactgccatacaa agtgtccaaa atcattaagc 300 ctgcattttt attattggga gtaatatcaaacctcctatt ttccaatttt catttcttgt 360 cctgtgctag ctccatcctg tttggactgctcctcccata tgtaaactaa gaagaatcaa 420 gcattctttg caacaaatac acacgatgctcaaaaatgtc caggagcatc caatttccaa 480 agtttcctcc acctggaatg ctcttcatgctaaaatcctg tctgacaata ccagcatctc 540 tggcctgcac tcatcccttc ctggaactccaagtgcattt accctctgtt accacttact 600 tggctgcctg aattgttagt tgaaaatattaggtctactt agctaattct tcctcaggaa 660 attaaagact cccatatggc agagtctgtgtcttttctct cttcatatcc cgtataacac 720 ccagcataat gctgggcata tagtgagtattccataaata gttgatgaat gactaaaata 780 agcaagcaaa caaacagact agaacaataagaaagaaggg actggatttc ataatctctc 840 tggcttgcta tttgaattgc tgaattattattatttatta aatatttttt aaattctggc 900 aataaaaggt aaggatttat tttctttctttctttttttt tttcttgaga cagagtctcg 960 ctcttactgc ccaggctgga gtacaatggcgcaatcttgg ctcacggcaa cctccgcctc 1020 ctcctgggtt taacagattc tcctgtctcagcctcctgag tagctgggat tacaggcata 1080 cgcccatgcc cggctaattt ttgtatttttagtagagacg gggttttgcc atgttggcca 1140 ggctggtctt gaactcctga cctcatgtgatccacctgcc tcagcctccc aaagtgctgg 1200 gattacaggc atgcgccacc gtgcccggccaaagatttat tttcaagaat gaaacaaagt 1260 aaggattctg ggtcaatctc acatgctgaaagccaaaacc tctagccgct cctgcttttt 1320 gacttcggag tgcccactat ctccgagcctgtgagcacag ggcctggcag aggggtttga 1380 gtggcatgag ctacctactg gatgtgcctgactgtttccc cttcttcttc cccaggcttg 1440 ttagagtgct gtgcaagatg tctggtaggggccccctttg cttccctggt ggccactgga 1500 ttgtgtttct ttggggtggc actgttctgtggctgtggac atgaagccct cactggcaca 1560 gaaaagctaa ttgagaccta tttctccaaaaactaccaag actatgagta tctcatcaat 1620 gtgtaagtac ctgccctccc acacagacccatcttttttt tccctctctc catcctggag 1680 atagagaact cttcagtacc ttagtaactagcaggggact ggggtggagc cagaccggat 1740 tcccgagtct tccctctgtg ca 1762 5828 DNA Homo sapiens 5 ctagaaaatc cctagccttg ttaaggtgct cgctctggtgtatacctcac ttatgtcggg 60 aaagaagcca ggtcttcaat taataagatt ccctggtctcgtttgtctac ctgttaatgc 120 aggatccatg ccttccagta tgtcatctat ggaactgcctctttcttctt cctttatggg 180 gccctcctgc tggctgaggg cttctacacc accggcgcagtcaggcagat ctttggcgac 240 tacaagacca ccatctgcgg caagggcctg agcgcaacggtaacaggggg ccagaagggg 300 aggggttcca gaggccaaca tcaagctcat tctttggagcgggtgtgtca ttgtttggga 360 aaatggctag gacatcccga caaggtgatc atcctcaggattttgtggca ataacaaggg 420 gtgggggaaa attgggcgcg agtctgtggc ctcgtccccacccaaggctg ggtcctctct 480 aggggcctgg catttgagtg aggaagcgat ggctgcagccgaacgagaag gtcaggaaga 540 acgtggtgcc cagctggctt agcctcacct ttcaaaggttccctaagcaa atttcttctc 600 aaaacagaaa gcatgagttt tgtgggatgc tttgtacaatcagaccattt ctaagccatc 660 tgttggtatc cctttgttcc cttcctagta ggtaccacaagagtggatct aactggacaa 720 gagtctaaaa tgctgctcat gtgattgaga cttgggcacctgagctraga gggaggatgg 780 ataataaaaa ttaaataata actccaaggt aaatttacaatgttctgg 828 6 1140 DNA Homo sapiens 6 gatcctcctc attcttcccc tacccattccccccaccctc cgttatactg gggccagtta 60 tctagtagat actgccaatt acccttggcagaggtgccct gctcactaat tttatttggg 120 ggagmgccct ggaacctggt tttaatgtctggcacacgcc acttccagga tctcccagtt 180 tgtgtttcta catctgcagg ctgatgctgatttctaacca acccatgtca atcattttag 240 tttgtgggca tcacctatgc cctgaccgttgtgtggctcc tggtgtttgc ctgctctgct 300 gtgcctgtgt acatttactt caacacctggaccacctgcc agtctattgc cttccccagc 360 aagacctctg ccagtatagg cagtctctgtgctgatgcca gaatgtatgg tgagttaggg 420 tacgggtgct ttggctctcc tacccactatggaagcacta tatatttggt tattttctta 480 gtgtaaggag ggtggtgatt atgagaaaaatataagatga tgaatgattg ggtcttagtt 540 tattaatcct tccctactga aaccagagaggtttcttccc ccggaaggga acttggaagt 600 ggtgggagtt ttcttggcca ttcacattggcctactctag ttgactgctg ttcacaaccc 660 caaagcagca catttcaata acaaacacaaggttdsacca ctgttcaata ccaccttctc 720 ttttttgtaa acctgtagaa aagaggatcctaattgttgg tagmatccaa mtttacagcc 780 aggataatta gagatggaag aagggctctgggggaaagtc tccatgtggc cccgtaactc 840 cataaagctt accctgcttg ctttttgtgtcttacttagg tgttctccca tggaatgctt 900 tccctggcaa ggtttgtggc tccaaccttctgtccatctg caaaacagct gaggtgagtg 960 ggttatttgg gttattttac aagggagtagctaataccat acaaattaca cccatggcct 1020 tcaattttaa ggactgaaag tttccctttgctggattttg aattagccga ttgccttcta 1080 caacatgttg gctaagtgtg cctgagccaatgagcataga aggtaaaaca cctcttttct 1140 7 295 DNA Homo sapiensmisc_feature (42)..(43) n at positions 42 and 43 is unknown 7 aattagcacacagaaaggat atccaacaca tacaaagctg tnntcatgga ctacactgga 60 gcatattactgctgttgcaa gaaacatttc ttcttcctct tttcattttc ctgcagttcc 120 aaatgaccttccacctgttt attgctgcat ttgtgggggc tgcagctaca ctggtttccc 180 tggtgagttgactttgaatg atcttggcaa gtaaataggc ctgagatagt tgtgggtaca 240 gctattctgaaaggcaagaa ggtagactgc ttccatcctt gaaatgctgg aggga 295 8 2940 DNA Homosapiens 8 aattctatat actatcacta tggctccact ttggatactc tccagtggatttagttactc 60 atatggaaat acctgggagg acctcctaac attattagaa ttgttatgattataatacaa 120 ygctatgtcc caggtcttgc tgatagtgct acagtgccct gtgaatgtagtgtgctcatt 180 gtgcagatta aaaacctaag gcactgaagg gtgaagtgat ttatctgaagttattttata 240 aagcagtgat cagacaasct gagctcacag aactccctgg cccctactgctgaggtttcc 300 atacagagtc aagtaatttc tcaccttgta aaacgaattg attcattaaccaggggagag 360 ctctactgca tgatgtggct gtgtgtctac agcaagcacc ctatgactctaagtcactcg 420 gacatattga tgtggcaaag cccaaatatt gttcacttcc ctgaggaaaactcagtgcta 480 gatcaaacag aggtgtggaa taaatcttta tgatttgatt ctctgggcctgggccatgag 540 acccatgatg cctcagagac atcggacttc cagtcaagtg tatatggagaaagccaagcc 600 tgggatgtac tgctttttgc agagcatggg tttttccctt atttagttatgattttattt 660 ctacccttcc tcattcccaa agggatttga ggagggagtg ctttcttttctactctcatt 720 cacattctct cttctgttcc ctacagctca ccttcatgat tgctgccacttacaactttg 780 ccgtccttaa actcatgggc cgaggcacca agttctgatc ccccgtagaaatcccccttt 840 ctctaatagc gaggctctaa ccacacagcc tacaatgctg cgtctcccatcttaactctt 900 tgcctttgcc accaactggc cctcttctta cttgatgagt gtaacaagaaaggagagtct 960 tgcagtgatt aaggtctctc tttggactct cccctcttat gtacctcttttagtcatttt 1020 gcttcatagc tggttcctgc tagaaatggg aaatgcctaa taatatgacttcccaactgc 1080 aagtcacaaa ggaatggagg ctctaattga attttcaagc atctcctgaggatcagaaag 1140 taatttcttc tcaaagggta cttccactga tggaaacaaa gtggaaggaaagatgctcag 1200 gtacagagaa ggaatgtctt tggtcctctt gccatctata ggggccaaatatattctctt 1260 tggtgtacaa aatggaattc attctgcgtc tctctattac actgaagatagaagaaaaaa 1320 gaatgtcaga aaaacaataa gagcgtttgc ccaaatctgc ctattgcagctgggagaagg 1380 gggtcaaagc aaggatcttt cacccacaga aagagagcac tgaccccgatggcgatggac 1440 tactgaagcc ctaactcagc caaccttact tacagcataa gggagcgtagaatctgtgta 1500 gacgaagggg gcatctggcc ttacacctcg ttagggaaga gaaacagggtcttgtcagca 1560 tcttctcact cccttctcct tgataacagc taccatgaca accctgtggtttccaaggag 1620 ctgagaatag aaggaaacta gcttacatga gaacagactg gcctgaggagcagcagttgc 1680 tggtggctaa tggtgtaacc tgagatggcc ctctggtaga cacaggatagataactcttt 1740 ggatagcatg tctttttttc tgttaattag ttgtgtactc tggcctctgtcatatcttca 1800 caatggtgct catttcatgg ggtattatcc attcagtcat cgtaggtgatttgaaggtct 1860 tgatttgttt tagaatgatg cacatttcat gtattccagt ttgtttattacttatttggg 1920 gttgcatcag aaatgtctgg agaataattc tttgattatg actgttttttaaactaggaa 1980 aattggacat taagcatcac aaatgatatt aaaaattggc tagttgaatctattgggatt 2040 ttctacaagt attctgcctt tgcagaaaca gatttggtga atttgaatctcaatttgagt 2100 aatctgatcg ttctttctag ctaatggaaa atgattttac ttagcaatgttatcttggtg 2160 tgttaagagt taggtttaac ataaaggtta ttttctcctg atatagatcacataacagaa 2220 tgcaccagtc atcagctatt cagttggtaa gcttccagtc atcagctattcagttggtaa 2280 gcttcccagg aaaaaggaca ggcagaaaga gtttgagacc tgaatagctcccagatttca 2340 gtcttttaat gtttttgtta actttgggtt aaaaaaaaaa aaagtctgattggttttaat 2400 tgaaggaaag atttgtacta cagttctttt gttgtaaaga gttgtgttgttcttttcccc 2460 caaagtggtt tcagcaatat ttaaggagat gtaagagctt tacaaaaagacacttgatac 2520 ttgttttcaa accagtatac aagataagct tccaggctgc atagaaggaggagagggaaa 2580 atgttttgta agaaaccaat caagataaag gacagtgaag taatccgtaccttgtgtttt 2640 gttttgattt aataacataa caaataacca acccttccct gaaaacctcacatgcataca 2700 tacacatata tacacacaca aagagagtta atcaactgaa agtgttccttcatttctgat 2760 atagaattgc aattttaaca cacataaagg ataaactttt agaaacttatcttacaaagt 2820 gtattttata aaattaaaga aaataaaatt aagaatgttc tcaatcaaacatcgtgtcct 2880 ttgagtgaat tgttctattt gacttcacaa tagaaactta ataatcgtaccttctcaaga 2940 9 17538 DNA Homo sapiens 9 atggaaatgt tctgtatttgtgttgtctga tgagataacc actaactgta gtgctattga 60 gcatttgaaa catggctagtgtaatcaatg aaccaaattt ttaattttat ttaattgtaa 120 ttaattttaa gtggccacatgcagggagtg actgctgcat tggacagcac ggctctaaat 180 tgagcctttt ttccttatttggtgaggcat acttgcctta agattgggaa gtctattttt 240 ggaacctgct accaatgctggtctcacact tgcaattctc agctgagcca agaggtgaga 300 gaaaggtcat tttccattccaagatctcac tctcccctgt gacactgagg aaactggcaa 360 gtgatgtgaa ggctggagagcgtgtcctgt atgctggctc tgtcccttct gcctgtgttg 420 actgacatag ttagttgctgcccttgctgg tctcccttcc tccaaccttg cctctctgag 480 cacacctgac attcatctcatgacttccct aaaaacattc tttgggaaca agaaactaac 540 aaatcccaag tgacctatcacatatacaaa catacagggc agagtttgga ttcgcggtag 600 aagaaaggga ggttagacattaagaagaat ggtctggtga tgacagttgt gagataatag 660 aaacaggaaa aagaaatctaagttttcttt ctttttttaa gaaccaataa taatttctct 720 cttttgacta gtcagtagggctggggtgga ttggaggaag cttacatatt ccatgaacaa 780 gcctcttcct aaggtcctgtaagtgatcct gccccactga ttagccccta gaagaccctt 840 caaaggttgg atctccaggagggagtgggg gaggaaagcc ctgtaccagg cagcctctgc 900 tccattgctc tgggggggtggggaagacaa accctggtca tcccctcagt ctgtagccct 960 tttgtgtgag tgcctggcaagggtgacgtg gggctgtttc tgcgggcaca gctgcagcaa 1020 ttaccggagt ggaggcagggcccaggcagc actgccctcc aagatcttcc cttgggcttt 1080 tcagcagtaa ggggacatgcaccccaaggg cctccacttg gcctgacctt gctgcggggg 1140 ctctctgtcc ccaggaacagtagagatggc aagcttatcg agaccctctc tgcccagctg 1200 cctctgctcc ttcctcctcctcctcctcct ccaagtgtct tccagctatg caggtaagac 1260 atgttttttt tcctgccctggggagaccct gaaaacagaa aggctagttt cctgggggtt 1320 agctccttca aacatcctcaagttggtata ttatctttct aaaacataga cctactgaca 1380 tgcctccctt cctcagaaaccttccgtggg tggttcttac agccttcaag atggagtcca 1440 gactcttttt tttttttgggacagagtctc cctctgttgc tcaggctgga gtgcagtggc 1500 atgatctcgg ctcactgcaacctcagcctc cctggttcaa gcgattctcc tgacttggcc 1560 tcccaagtag cggagactacaggcgcctgc caccacaccc agctaaattt gttcttttct 1620 ttcttttttt ttttttttgggattttagga cagacggggt ttcacatgtt ggccaggatg 1680 gtctcgatct cttgacctgctgatccgccc gcctcagctt cccaaagtac tgggattatg 1740 ggcgtgagcc actgcactaggcctaatttt tttattttta gtagagatgg ggtttcacca 1800 tgttggccag gctggtctggaacccctgac ctcaagtggt ctgccctcct cagcctccca 1860 aagttctgag attacaggcatgagccattg cgtctgaccc agactcctta atgtgactaa 1920 ctccaggctt tccttggactacttcttact tgtctttcca gctttgtctt ttcacctctc 1980 caattgagat aaaataataacaacctcttg gagttctcat caggattaca tgaaatgaga 2040 tatgtaacat gcttagcagtgcctgtccat agtaaatctc aataaatgtt tgtggaatta 2100 taatatcttg tcatgtttgagactttgctc tgcataatca ggcaccagta ggtttttata 2160 aaggaacccg tctgtcacgtgcagaggaga aataaacaga aagtttccca tcctcaggga 2220 gccacctgac tgacagaggcacagtgcatc cactctccag gtctagggga gaaagcagcc 2280 ttatttctta gtagctcagaatctgacttg agaaacacat ccacatagaa aaaaacaagg 2340 aactttttcg ggtcagggtccgggacccac agtgaggtgg aagatacagg ggaaggaaga 2400 gggaaataga gccatccccagggtggaaga tctcagaaga gaatttggga aacaaggtat 2460 gaacaaggac tgaatagtgagaagtgatgg agagacagct aaagtagatg gagtgtcaaa 2520 accaaaacct ctaagggtagaataggcagc aatttggcca agtcctaaca gggaggccca 2580 taggaggatt caacctcaagatgctgtgcc acattccaag agggaaccta aaggctgggc 2640 tgaagagtca gagatggctacagctggcaa aaagatgggc agatgctgag aggagatgat 2700 tgctaaaatg ttctgtccaggacattcaca gtatctctat aaccagagtc ttttttgtcg 2760 ttgttgttct caagaaggaaacttgaggcc gggtgtggtg gtttatgccc ataatcccag 2820 cgctttgggg ccaaggcaggcggatcacct gaggtcagga gttcgagacc agcctggcca 2880 acagtgtgaa acctcatctttactaaaaat acaaaaatta gctggatgcg gcggtaggtg 2940 cctgtaatgc cagctactcgggaggctgag gcaggagaat cacttgaacc tgggaggcgg 3000 aggttgcagg gaggcggaggttgcagtgag ccaagattgc accactgcac tccagcctgg 3060 gcgacagaga gtaagactgtctcaaaaaat aaatgaataa ataaaaagga agaagaagaa 3120 gaagaacaat tgcaatcctccctggctcta gaatgtcatt taaaagtcga gtgtcttctt 3180 ccttccctgt tttgaagcagcccttctcat gacaggcttg cttgccaagg ttccctctga 3240 ccttaaatct cttccttttggtgtcttgga cagggcagtt cagagtgata ggaccaagac 3300 accctatccg ggctctggtcggggatgaag tggaattgcc atgtcgcata tctcctggga 3360 agaacgctac aggcatggaggtggggtggt accgcccccc cttctctagg gtggttcatc 3420 tctacagaaa tggcaaggaccaagatggag accaggcacc tgaatatcgg ggccggacag 3480 agctgctgaa agatgctattggtgagggaa aggtgactct caggatccgg aatgtaaggt 3540 tctcagatga aggaggtttcacctgcttct tccgagatca ttcttaccaa gaggaggcag 3600 caatggaatt gaaagtagaaggtgagtagt gccatataat attaggtatt aactgttggg 3660 tggccaagaa caattattctctcaactgag atgagatccc tcaacccaaa catctcagtc 3720 ctgggaatga tttccataaaaatgtacaca tcaataaaca gaaactcatg cttagggatg 3780 tctgttgcat cattattcagagtagcaagg aaattgggat caaaatcaat gcctttgagt 3840 aggtaagtga cagaatgaacaatggtagcc atactgtgaa tattatgcag ggattaaaaa 3900 gattatttta gcactaggccagatggtttg gggggctcct ctaaggtatt attgagtgat 3960 aagagcaagc tgctgtaggatacaaaaaca aaaacaaaac cctagggcat ggtggtttgc 4020 ctcgcagcta ctcaggaggctgagacggga ggctggcttg agcccagggg tttgcagtta 4080 cagtgagcta tgattgcaccactgcactcc aacccgggtg acagagcaaa gaccttcacc 4140 cccactccct acccgtctctaaaaaaaaca aaaacaaaaa caaaaaaacc cttgggccca 4200 gcgccgtggc tcacgcctgtaatcccagca ctgtgggagg ccgaggtggg cagatcacaa 4260 ggtcaggaga tcgagaccatcctggctaaa acggtgaaac cccgtctcta ctaaaaatac 4320 aaaaaaaaaa aaaaaatttagccaggcatg gtagcaggcg cctgtagtcc cagctactcg 4380 ggaggctgag gcaggagaatggcgtgaacc cggaagcgga ggttgcagtg agccaaaatc 4440 cttccactgc actccagcatgggggacaca gcgagactcc gtctcaaaaa aaaaaaaaaa 4500 accctgtatt tgtgagcgcacacacacaca cacacacaca cacacctgtg cttggtccta 4560 gtgaataagc aagtaaatcaaatgtctaaa tataattata gaaaggagat gtcacctttt 4620 ggctgtacct ccactatttcattctgcaga attgcagaat ttcttttttt tttcctttct 4680 ttcttttctt tttttttttgacacagagtc tcgctctgta acccaggctg gagtgcaatg 4740 gcgccctccg cctcctgggttcaagtgatt ctcctgcctc agcctcccga gtagctggga 4800 ttacaggtgc ccaccaccacacccagctaa tttttgtatt tttagtagag acagggtttc 4860 accaggttgt caaggttggtctcaaactcc tgacctcagg tgatccactc gcctcagact 4920 cccaaagtgc tgggattacaggcatgagcc atggtgcccg gcctcagaat ttcattttca 4980 acatgttttg catgatgggtgattttggag aatatttttt gctctatcgc aggatgatta 5040 agatgtggac aaggtgaagccgatggaggg ggagctttga aagttacttg ctatttaatt 5100 gaggaactaa actgctttgagagcctgggg gtcagatcct ctgccttttc ctcctcccca 5160 cctgcagtgc aaacatcagacaattgatca ctattgtatc ttggaggtgg gagtgaccat 5220 tgcagtgctg ggaccagaagatggcattgt atgtggaaca acaaagcact atttctagag 5280 actgcctgca gggatatggaaatagcttta tgtgtctcag aatgttcttc atacagctgt 5340 ttttattggg gaaattctacttgccgaaaa gtttgatagt gagaccctct ccagtttgca 5400 gatttttctc cttcctgctcaacaacttcc tagctcagta actgcctctc ccaacaaact 5460 ccctcagttt caccacaccaaaaaaggaag acaagccggt tgcggtggct cacacctata 5520 atcccaaaac tttgggaggccgaggcgggt ggatccacct gaggtcggga gttcgagact 5580 agcctgacca acatggagaaaccctgtctc tactaaaaac acaaaattag cctggcgtgg 5640 tggcgcattc ctgtaatcccagctgggagg ctgaggcagg agaatcgctt gaaccccgga 5700 ggcggaggtt gcagtgagccaagatcgttc cattacactc cagtctgggc aagaaaagtg 5760 gaactccatc tccaaaaaaaaaaaaaaaaa aacaaggaag acaaaaagaa aagcagctaa 5820 agactttgcc tcaggggagaaagttctctt ttgggttgct atccacattc caacctcctg 5880 ttcccacctc ttcgtctgcatgcctaagaa actgttttac aagtaaataa gggacgcttt 5940 gtctaggctt tggagccaggaagttgagac aaatttagga atgagatgaa gtaatggtat 6000 tattgcaagt ctcaggtgtaactacctctg ctctttctct gaagagtttc taatttctct 6060 tgtttactta tttttttcttgtcatttttg ggattttatt actagttgtc tctaatcctt 6120 tctttaaatt cttcattatgaaacataaaa acaaatgcca ggcgcggcag ctcacgcctg 6180 taatcccagc actttgggaggccgaagcgg gcagatcacc cgggtcagga gttcgagacc 6240 agcctgatca acatggagaaaccccgtctc tactaaaaaa tacaaaatta gctaggcgtg 6300 gtggcacatg ccagtaatcccagctacttg agagactgag gcaggagaat cgcttgaacc 6360 gggaggcaga ggttgcggtgagccaagatc gcgccattgc actccagcct gggcaacaag 6420 agcaaaactc tgtctcaaaaaaaaaaaacc acatacaaac cagagataat attataatga 6480 gcctccaagt gcctaccaccttgctgcagc acttgtcaat ccagggacca cccacctcac 6540 cggctcccca ctcattaccaccctccccta ctcaattact gaggtaaatc ctaggcagca 6600 tgatcatttc ttttttttctttttatttat tttgagacag gatctgtctc tgtcacccag 6660 gctggagtgt agtggcatatctctgctcac tgcagcctct gcctcccggg cagaagccat 6720 cctcccacct cagcctacatagtagctggg accacaggca cacaccacca cacactgcta 6780 atgttttgta ttttttgtagagactgggtt ttaccatgtt gatcaggctg gtctcaaact 6840 cctaggctca agcaatcctcccacctcggc ctcccaaagt gctagaatta caggcgcgag 6900 ccactgcacc cagcgaagaacactttttaa aaaataaata ggccgggcgc ggtggctcac 6960 acctgtaatc ccagtactttgggagcccaa ggagggcgaa tcatgaggtc aagagattga 7020 gaccatccta agtaacatggtgaaacccca tttctactac aaatacaaaa acaaaattag 7080 cctggcgtgg tggcaggcgcctgtagtccc agctacttgg gagctgaggc aggagaatgg 7140 agtgaacccg ggaggcggagcttgcagtga gctgagatca tgccactgca ctcccccctg 7200 gggcaacaga gtgagactcccaaaaaaaaa aaaaaaagcc ccccctcccc acacacaata 7260 atataaataa ataaataaccacaatactat tatcacatct tacaaactca acaaaaattt 7320 cttaatatca tcaaatacccagtttgtgtt caaattttcc tgattgtttc ataaatatac 7380 tcttacagtt ggtttcttttagcgagattc aaatgagacc cacctgttga cctttgccct 7440 tagggtttcc cagggtctgaattttgttga cgacattccc atgttgctat gtaatacggt 7500 cctccatgcc ctgtgtttttctgtaaactg atagatgtgg aggtgcaatg acatttgtgt 7560 ttgatttact ttggcaaatatagttcatca gtgatactct atacttcttg ttgctttaca 7620 tccggaggct gataatgtctgcttttctct cttttctaat tatttgtgaa aggaaaaatg 7680 tggggggttg ggagaaaaaaacccttaagt acatactcgc taaatcacat tgctacaggt 7740 aacttccatt aagaacttgaaagtaaaggt agctgcattt tcccctaggg aacacaatga 7800 tagacaggag ccttagtctacagcttgaag gattgtaatt atacctaagc aaccctcctg 7860 gaccagttta atgttattagctgtgatgta tccctacctt tgatgtcatt atccttactt 7920 agctccctta aagcagagatcaagatgaaa agggcttcag ctgcagcatg gcacatggag 7980 attagagtgg ggcttttggatgctgaggag cagacctaga atgggaaata gatgggagcc 8040 acagaagtga aggtccccctccctcattgc tcaacctact ccacatctcc aggtctgcac 8100 atctgttcag ttactgaatcctgtgtaagc taccttcttt ttcttttttc ttttatttat 8160 ttatttattt tttttttgagatggagtttt gctcttgtta cccaggctgg agtgcaatgg 8220 tgcaatctcg gctcactgcaccctccaact cccaggttca tgcaattctc ctccctcagc 8280 cttccaagta gctgggattacaggctgcac caccatgtct ggctaatttt tgaaaaatca 8340 gtagagagag ggtttcaccatgttggccaa gccggtctcg aactcctgac ctcaagtgat 8400 ccacccacct tggcctcccaaaatgctggg attacaggtg tgagccacca tgcccgctgt 8460 aaactacctt cttaaaagctctagaagagg gcttttaacc ttttgttgtg tgtcatgcac 8520 cttccgcaag ctgatgaagttgatagaccc atctcagaat tttttttttt tttttgagac 8580 agtgtctcac tctgtcacccaggattggtt gcagtggcac gatcatgggt cattgcagcc 8640 tccacctccc aggctcaagtgatcctcctg actcagcctc ttgaatagct gagaccacag 8700 gcttgtgtca ccatgcccaggtaattttta attttttttc gtagaggcag ggtctcacat 8760 tatgttgccc agtctggcctcgagaactcc tgggctcaag caatcttcct gccttgggct 8820 cccaaagtgg tgggattacaggggagagcc accacaccta gccaggagga tgttttaaat 8880 acaccaaata aaacatttatacccaaatac agttatccaa atattaaatt aacaagagtt 8940 agggtgaccc tattaattagtgtaatttcc aaatagtaat gaacataagt gatagtttga 9000 gatttctgtg acttttctaatgtgacgtga aaatatttgt gatttttctt tttctttttt 9060 ttttttgaga tggagtttcgctcttgttgc ccaggctgga gtgcaatggc aagatctcgg 9120 ctcacctcaa cctccgcctcctgggttcaa gcgattctcc tgcctcagcc tcttgagtag 9180 ctgggattac aggactgtgccaccacgtcc agctaatttt gtatttttag tagaaacagg 9240 gtttctccat gttggtcaggctggtcttga actcccaacc tcaggcgatc cgcccgcctc 9300 ggcctcccaa agtgctgggattacaggtgt gagccaccgc acctggccaa tatttgtgat 9360 ttttattgac gacaaagtcaaaggttctct tcatattatt gtggtgtatc gcctacaagc 9420 ataattaaaa taaacactaaatttcagttt aaagtttact gaaaataaat atgtattttt 9480 tattccctat ttaagctttgaatcccctga cttcctatac cattaccact gtcctagttc 9540 aggttcatgt tgttttttactttaattgtt atcacagtct cttaacattt ctccctatgt 9600 tctccagtcc tgtaggtgctaaatctgacg tggtcacttc tcagcttgga atccttcagt 9660 gcaccaccac agccttgaactacatatttg aaatacatat ttattttcag taaactttaa 9720 actgaaattt agtgtttattttaattatgc ttgtaggcga tacaccacaa taatatgaag 9780 agaacctttg actttgtcgtcaataaaaag tcccttgagg ggacttcaga tgtaagtccc 9840 ttagctgctc gttaaaactcccccaggctg acccaataca caatcttgac tttaaaccac 9900 ttgtcattct aaatcactagcatttcctgg aaaaaaaagc catttttcct tcagggctaa 9960 gctcagggac caattctgtgtcaccttctt tgaatcctga tgatattcac ttctttattt 10020 gacctgattt attgggccccagacaccatg ctgagtgttg gggattcagc tctggacaat 10080 gtcaaatgtc agtcctgcctttcagatcct ttctactggg tgagccctgg agtgctggtt 10140 ctcctcgcgg tgctgcctgtgctcctcctg cagatcactc ttggcctcgt cttcctctgc 10200 ctgcagtaca gactgagaggtacagggcag agggtgggtg gatcaggatc ctttctttaa 10260 atgagctggc ttcttggagctacaccactt aacatgtatt tgtgagtgac ttctgggttc 10320 agaagttctt ctcactattgagtgataaag aaaaaaaata actccatgat gaaagagttt 10380 tacatcttac ggaatgctttcatatgaata atcggaccta gcatttccct atgagctaac 10440 tatgccatat agtaaccccattttacagag gatacaactg aggccaggag tagttcagtg 10500 acttactcaa accgatataacttataagtg gtagagctga ggcctctgta tcatacctag 10560 cagctccatg caacttgggagagtgtgagc ttcgaagtca gacaggtcta ggctattagg 10620 agttttgaat aaagatactgaagtgaaagt ctctaccaca cagtaggcgt tcgaaaattg 10680 tttcctcttt ctccattcaacactgaggac tcaggttcag ctgctgatga agctcctctt 10740 ttttgcctag agctttcattctgagccttc tcctcctacc aagtgtctcc ccaatgccag 10800 agcaggaaga gtcttcactcctcccaatgc cccacctccc atttgttact aagaggagag 10860 gagaaagtag caaggagggtatggggaatg ttctggggga atgggtgttg gtgcgatcaa 10920 caacaaagtc ctttctctcaccttgaattc atcccagatg cctgcttgtt tacttcttcc 10980 acacaaaaaa aggccttcagccctcatggc tgagcagaaa gaatctgaat gttagagtca 11040 ggcagcctgg gtttgaattccatctcaggt actgaactct atagcaaaat tcttagattc 11100 tccaagcttc agttgccttgtctgtcaaat agagaaaaca tccttcgtcc taaattgtag 11160 ggaggattaa agtcatgcaaagtgcctact acaaatccag tcacaaagta gctagctact 11220 cactaaatgt tcagctcctccctcctcatt cagatgggaa gtggctttag ataaacaaag 11280 tggcaacgca gtgggctggagcagctctgt gaactgagaa tccaagaaaa ggggcgaaga 11340 gcagctggga tgtattggatgcttgtgctg gcttggagca ttgctcacat tctttattcg 11400 ctattgtatc tagactatagctagagaaag agccgcaacc attggcttta aatccagtgc 11460 tcttcctact ctcctgaggttgtttccagg ctgcagagaa atagcctgca caaggggccc 11520 aggcgctggg tgtgggagggtccccaccga gagccagaac atgcaggaac taaaatgttg 11580 cctttttcta ttttaggaaaacttcgagca gagataggtg agttccagtc atcgtttctc 11640 ccaattcttg ccttttggttttttggcata acggaaatgg tcccattctt ggaccgtctc 11700 tccctctcaa taccctgttttcccctcagt ttccctttct ctacagtggg tgtgtcgtgc 11760 ctagaacaag ttttaagtaattaaataaca aagactcagg ataaaaggat cctttttgga 11820 gtgccctact aaatccatttccatttgttt ctctttcaga gaatctccac cggacttttg 11880 gtaagttccg gcatgtctaggccctcccag gtcaacttgg tatttcactc tagttccagt 11940 cacctggggg aacaaggacccctggctcct ggttgagtcc cttcctctct tctcttttct 12000 ttctttaaat aagaagtcatttgcatttag gattggtaaa atcataataa aaatactcat 12060 gtactgtttt tatgtgccaggcactattct aactacttta caaaaacgtt atcttattct 12120 gtttaactcc ttatgcacatgatctctctt ttcaggaatg ccaaaacaga ggtaaataga 12180 tcgtttacac gtaaacctgatgtctggttg gggaggtgaa acaaacagaa acaagacaca 12240 actgtatcac ctgtacttatatttctgctt tacaaactca ggatgtttcc atgagtacag 12300 aacatgacta atcagagaagacctcataga ggaatagaaa agccaccaag ccccactagg 12360 aattgacccc tcaaggacatggtttctagc ctttttgttc actgcagatt gcccaatgcc 12420 taaagataat ggcaacagaagagcacccaa atatttgtta gataaatgtt gcagacacta 12480 gaaggtgtca ttagggcacagatggtacct tctctgagca aacttccttc acagctcctc 12540 ctcccgaggc tgtaggtgactctactcttg tcacctggca cacagagttc tatcgtacga 12600 tttaggaaat tagaccagtgtgtggaccac acacacacac atctttacac acccaaagag 12660 gaggaatagt atctttgttttggaggactt gactatgaaa ggtcttaact cctttttgta 12720 ccatgaatct ctctggcactccagtgaagt ctaaaggacc cctttgcaga atgtttttaa 12780 atatacacat aaaatagaacacataggatt gcaaaaacaa tcattgtact aaaatacagt 12840 tatcaaccga taatcacatttgtgatatag taacataaat gtttcttttt tttttttttg 12900 gaggcagagt ttggctcttgtcacccaggc tggagtgcaa tggcgcgatc taggctcact 12960 gaaacctctg cctcccgggttcaagcgatt ctcagcctcc tgagtagctg ggattacagg 13020 tgcccgccac cacacccagctaatttttgt atttttagta gagactaggt ttcaccaggt 13080 tggccaggct ggcctcgaactcctgacctc aggtgatcca cctgccttgg cctcccaaag 13140 tgctgggatt acgggcatgagccaccgtgc ccggccataa atatttcttt agccaaagta 13200 atacattaag taatgtagcagcaagtctaa taacctgtaa tttctttctt tctttctttc 13260 tttctttttt tttgagatgaagtttttttg agatggagtg caatggcaca atctcggctc 13320 actgcaacct ccacctcctgggttcaagcg attctcctgc ctcagcctcc caagttgctg 13380 gaactacagg cgcatgccaccatgcccagc taatttttgt atttttagta gagacggggt 13440 ttcaccatgt tggccaggctggtcttgaac ccctgacctc aggtgatctg cctgccttgg 13500 ccttccaaag tgctgggattacaggcatga gccaccaggc ccagcccaat aacctttaat 13560 ttcaacatac taataaacataaacagtatt tcaagatttc tgcaataact ctaatgggaa 13620 tgaaaacatc tgtggcttccattggtaatt aagtcacagg tactgctcat attgtggtta 13680 gttgtaaaat gttttggtttgttttgtttt ttccaagact tgggggaatg ggtgttggtg 13740 ggatcaacaa gagtcttgctctgtggccca ggctggagtg caggggcagg atcttggctc 13800 actgcaacct ccgcctcccaggttcaagcg attctcctgc ctcagcctcc tgagtagctg 13860 gcattacagg catgtgccaccacgcccagc taatttttac atttttagta gagatggggt 13920 ttcaccatgt tggcctggctggtcttgaac tcttggcctc atgatccacc cgtctcggac 13980 tcccagagtg ttgggattacaggcatgagc caccacacct ggcagttgtt acatttttaa 14040 tgaaagaaaa tgttaaatccagttattgaa aataaggagg cagtactttt ctcatccaag 14100 ttcatggact ttctgaattttgtccccaga gtcctttggt gttctaggac cccaggttaa 14160 ggaacccaaa aagacaggtgggtggggcat gagggggaac acatgttaat ccctgtttgt 14220 tctggtgaac aattcagatccccactttct gagggtgccc tgctggaaga taaccctgtt 14280 tgtaattgtg ccggttcttggacccttggt tgccttgatc atctgctaca actggctaca 14340 tcgaagacta gcaggtgcagtggctgggca gcaggcaaga ccaccaaata gtgggggacc 14400 aagtcagctc tgaatgggaagccaaaagag aatagaacca ggactcaaga ttaggggagc 14460 tgggatttcc ttattcctctgtccccatgc ccaaccccag gctcttctga gaaactgtga 14520 agagaaccac ttactggatctgtgggatcc cccagtggaa agggcagtgt gggtcactcc 14580 aaatgtccat agggaggatgtggggaaggt gctattcatc ttccactaat cacatatttg 14640 tttctttttg ttttcagggcaattccttga agagctacgt aagttctctt ctctctgtta 14700 taagcagaga ataaaaagccaggaaaggga gacagaagca acaagaggaa gaggcgggct 14760 attgagggat cacattcccagaggaaagga ggagctggag agcctgggtg gagggaagac 14820 tcctcctggg aggtagagggcaaagaagcc agctgttaga gacacattta caggtggcag 14880 agaagctgga ggcactcctatctgccacct gatccattcc tccttcactg cccctaagca 14940 ggaatccaac cctagctggtctcattgccc attccacagc aactgcccag tgcctcacct 15000 ctcagatcaa ccattgaggcaggaatggag acaagatgac cccaagggct tttcttctcc 15060 ctagttcaat ggttttatgatacaaactac tgacatacgt ttttcaagtt attttctcct 15120 tcttctagga aatcccttctgagtgatgtc acatcttggc aggggtggag gagagcctgg 15180 ttgcccaggg atttgtccttggggacatct catccatcaa gttgcacact cactggcatc 15240 tttgctatgg ggacattccaatttgcactt tcaggaacac tctgaattcc aagtagaatt 15300 gatttccctt cttctgtcatctaccttttc tcttcatttt cccattttta ttacccttct 15360 ttccatttct ctctccagtcttccacctgg aagccctctc tggctaagga caggcaggtg 15420 cccctctctc catcagaggacacctgtact ggagagcaac acaggatggt ctctgccatg 15480 aactggaggc caggaatctcctcactgaaa attacagtat ggtaactttg caaatggtgg 15540 ttgtttcttc caagactccagccctgattg cgcaaaactg aaaggcatgt gaagggaagg 15600 aagaggaaga gtgcaaaacattgaagagag agctgagtga gctgaagagt gaggatatga 15660 gtagccccaa cccaaacctggagatgggga gaaacctaca gaatactagc cagagctcct 15720 ccttgtcttg gcagcctactagggacctgg ggaagcaaaa acgaaagctg ggcaacatgc 15780 ctgctttaga atgttttccttctacttaca catcttccac aggtctcaga atctttcctt 15840 cctctcatcc ttttctcctatctacatatc tatcagagta tccactgttt attcaacaac 15900 tactacttga tggtcagacacaaacaaaca agctaggtgc taattaataa agatacgagt 15960 tttggccggg tgcggtggctcacgcctgta atcccagcac tttgggaggc cgaggcgggc 16020 gaatcacgag gtcaggagttcaagaccagc ctggccaaca tggtgaaacc ccatctctac 16080 taaaaataca aacaattaactgagcatagt ggtgggcacc tataatacca gctactccgg 16140 aggctgaggc aggagaatcgcttgaaccca ggaggcagag gttgcagtga gctgagatcg 16200 cgccactgca ctctagccggagtgacagag taagactctg tctcaaaaat aaataaataa 16260 ataaataaat aaataaataaataaataaaa aataataata caagttttca taagcacact 16320 tctaacccct tgtcttttatgtatttcctt ccttatccac gcacctgtct ccctctactc 16380 cagcctcatt accccagaggtcagtcctca ggaaaactaa acacaaagaa agagctcagt 16440 cagaaaggcc atttatttatgtttcaagat gctcactgcc tcctttgttt tgtctccttt 16500 gcaggccttc tctcttaggcctcttctcct gggggtatgg atcctggggg gagattgatc 16560 acctccatgc ttccattcctccccagccat agtggggaca tcatgagaga agccaagcca 16620 ctggcccagg atcacccggcatttatggtg gctgctctgg cacaggtcct tgcctttata 16680 gcccctccag tgatccataaggccctcttt ctccccaaag gagaggtcac agatagggca 16740 aaggtagctc ttctgcttccagtgggtctg ctggtgtctg accagcctgg aaaatgagct 16800 gaaagacttg ctgcaatggaagcagtagtt gggcggctct gtgaggtggc ccttctggtg 16860 tctggagaga taggatttcttgctaaaagt caaagaacaa tgggggcaac agaagacatt 16920 gagtcttgag ggcttcactggatgagagtt ggatctggca tcctgacaga gggttccagt 16980 gatgggtgcc tgggtcctggtcacaggtgc ttggttctta agtacagatg cctggttctg 17040 ggccatagga ccctcagttctaaatatggg ttcctgggac ctggccactg gtgcatggtt 17100 cacatccaaa agcccctggatggacctctg gcttctggcg atgggtgtct ggaattcagc 17160 ctgggtgcct ggaatcctcaaagtacactc ctggtttcca tccactggct cctggttttg 17220 gtgtatcttc tggtggcgtttgagctcaga ctggtcccgg aagctcttcc cacacacaga 17280 gcatgaatgg ggccggtaacccagatggac gcggcggtga cgacttagtc cagaagcatc 17340 acagtaggtc ttgtcacagagcgtgcaaca gaagggcctc tccccaagat gcatgcgtct 17400 gtgatagctg agggacttggggctccgaaa caacttccca cactgactgc agctgttagt 17460 cagcttggga ttgtgaacaaactggtggct atagaggtag gagcgcctgc tgaaacattt 17520 ggcacaggtg tagcaaaa17538 10 327 DNA Rattus norvegicus 10 tttgtatgtc attgcaggat tcatgctttccagtgtgtca tctatggaac tgcctctttc 60 ttcttccttt atggggccct cctgctggctgagggcttct acaccaccgg cgctgtcagg 120 cagatctttg gcgactacaa gaccaccatctgcggcaagg gcctgagcgc aacggtaaca 180 gggggccaga aggggagggg ttacagaggccaacatcaag ctcattcttt ggagcgggtg 240 tgtcattgtt tgggaaaatg gctaggacatcccgacaagg tgatcatcct caggattttg 300 tggcaataac aaggggtggg gggacaa 32711 2013 DNA Rattus norvegicus 11 ctgtatcagt gctcctcgtc gcctcactgtacttcacgga agagacttgg ttgactggcc 60 acttggagcg gaatcaggag acattcccaactcagagaga ctgagcccta gctcgcccac 120 ttgctggaca agatgatatt ccttaccaccctgcctctgt tttggataat gatttcagct 180 tctcgagggg ggcactgggg tgcctggatgccctcgtcca tctcagcctt cgagggcacg 240 tgtgtctcca tcccctgccg tttcgacttcccggatgagc tcagaccggc tgtggtacat 300 ggcgtctggt atttcaacag tccctaccccaagaactacc cgccagtggt cttcaagtcc 360 cgcacacaag tggtccacga gagcttccagggccgtagcc gcctgttggg agacctgggc 420 ctacgaaact gcaccctgct tctcagcacgctgagccctg agctgggagg gaaatactat 480 ttccgaggtg acctgggcgg ctacaaccagtacaccttct cggagcacag cgtcctggac 540 atcatcaaca cccccaacat cgtggtgcccccagaagtgg tggcaggaac ggaagtagag 600 gtcagctgca tggtgccgga caactgcccagagctgcgcc ctgagctgag ctggctgggc 660 cacgaggggc taggggagcc cactgttctgggtcggctgc gggaggatga aggcacctgg 720 gtgcaggtgt cactgctaca cttcgtgcctactagagagg ccaacggcca ccgtctgggc 780 tgtcaggctg ccttccccaa caccaccttgcagttcgagg gttacgccag tctggacgtc 840 aagtaccccc cggtgattgt ggagatgaattcctctgtgg aggccattga gggctcccac 900 gtcagcctgc tctgtggggc tgacagcaacccgccaccgc tgctgacttg gatgcgggat 960 gggatggtgt tgagggaggc agttgctgagagcctgtacc tggatctgga ggaggtgacc 1020 ccagcagagg acggcatcta tgcttgcctggcagagaatg cctatggcca ggacaaccgc 1080 acggtggagc tgagcgtcat gtatgcaccttggaagccca cagtgaatgg gacggtggtg 1140 gcggtagagg gggagacagt ctccatcctgtgttccacac agagcaaccc ggaccctatt 1200 ctcaccatct tcaaggagaa gcagatcctggccacggtca tctatgagag tcagctgcag 1260 ctggaactcc ctgcagtgac gcccgaggacgatggggagt actggtgtgt agctgagaac 1320 cagtatggcc agagagccac cgccttcaacctgtctgtgg agtttgctcc cataatcctt 1380 ctggaatcgc actgtgcagc ggccagagacaccgtgcagt gcctgtgtgt ggtaaaatcc 1440 aacccggaac cctccgtggc ctttgagctgccttcccgca acgtgactgt gaacgagaca 1500 gagagggagt ttgtgtactc agagcgcagcggcctcctgc tcaccagcat cctcacgctc 1560 cggggtcagg cccaagcccc accccgcgtcatttgtacct ccaggaacct ctacggcacc 1620 cagagcctcg agctgccttt ccagggagcacaccgactga tgtgggccaa aatcggccct 1680 gtgggtgctg tggtcgcctt tgccatcctgattgccattg tctgctacat cacccagaca 1740 agaagaaaaa agaacgtcac agagagccccagcttctcag cgggagacaa ccctcatgtc 1800 ctgtacagcc ccgaattccg aatctctggagcacctgata agtatgagag tgagaagcgc 1860 ctggggtccg agaggaggct gctgggccttaggggggaac ccccagaact ggacctcagt 1920 tattcccact cagacctggg gaaacgacccaccaaggaca gctacaccct gacagaggag 1980 ctggctgagt acgcagaaat ccgagtcaagtga 2013 12 171 PRT Homo sapiens 12 Met Ala Ser Gln Lys Arg Pro Ser GlnArg His Gly Ser Lys Tyr Leu 1 5 10 15 Ala Thr Ala Ser Thr Met Asp HisAla Arg His Gly Phe Leu Pro Arg 20 25 30 His Arg Asp Thr Gly Ile Leu AspSer Ile Gly Arg Phe Phe Gly Gly 35 40 45 Asp Arg Gly Ala Pro Lys Arg GlySer Gly Lys Asp Ser His His Pro 50 55 60 Ala Arg Thr Ala His Tyr Gly SerLeu Pro Gln Lys Ser His Gly Arg 65 70 75 80 Thr Gln Asp Glu Asn Pro ValVal His Phe Phe Lys Asn Ile Val Thr 85 90 95 Pro Arg Thr Pro Pro Pro SerGln Gly Lys Gly Arg Gly Leu Ser Leu 100 105 110 Ser Arg Phe Ser Trp GlyAla Glu Gly Gln Arg Pro Gly Phe Gly Tyr 115 120 125 Gly Gly Arg Ala SerAsp Tyr Lys Ser Ala His Lys Gly Phe Lys Gly 130 135 140 Val Asp Ala GlnGly Thr Leu Ser Lys Ile Phe Lys Leu Gly Gly Arg 145 150 155 160 Asp SerArg Ser Gly Ser Pro Met Ala Arg Arg 165 170 13 274 PRT Homo sapiens 13Met Gly Leu Leu Glu Cys Cys Ala Arg Cys Leu Val Gly Ala Pro Phe 1 5 1015 Ala Ser Leu Val Ala Thr Gly Leu Cys Phe Phe Gly Val Ala Leu Phe 20 2530 Cys Gly Cys Gly His Glu Ala Leu Thr Gly Thr Glu Lys Leu Ile Glu 35 4045 Thr Tyr Phe Ser Lys Asn Tyr Gln Asp Tyr Glu Tyr Leu Ile Asn Val 50 5560 Ile His Ala Phe Gln Tyr Val Ile Tyr Gly Thr Ala Ser Phe Phe Phe 65 7075 80 Leu Tyr Gly Ala Leu Leu Leu Ala Glu Gly Phe Tyr Thr Thr Gly Ala 8590 95 Val Arg Gln Ile Phe Gly Asp Tyr Lys Thr Thr Ile Cys Gly Lys Gly100 105 110 Leu Ser Ala Thr Val Thr Gly Gly Gln Lys Gly Arg Gly Ser ArgGly 115 120 125 Gln His Gln Ala His Ser Leu Glu Arg Val Cys His Cys LeuGly Lys 130 135 140 Trp Leu Gly His Pro Asp Lys Ile Thr Tyr Ala Leu ThrVal Val Trp 145 150 155 160 Leu Leu Val Phe Ala Cys Ser Ala Val Pro ValTyr Ile Tyr Phe Asn 165 170 175 Thr Trp Thr Thr Cys Gln Ser Ile Ala PhePro Ser Lys Thr Ser Ala 180 185 190 Ser Ile Gly Ser Leu Cys Ala Asp AlaArg Met Tyr Gly Val Leu Pro 195 200 205 Trp Asn Ala Phe Pro Gly Lys ValCys Gly Ser Asn Leu Leu Ser Ile 210 215 220 Cys Lys Thr Ala Glu Phe GlnMet Thr Phe His Leu Phe Ile Ala Ala 225 230 235 240 Phe Val Gly Ala AlaAla Thr Leu Val Ser Leu Leu Thr Phe Met Ile 245 250 255 Ala Ala Thr TyrAsn Phe Ala Val Leu Lys Leu Met Gly Arg Gly Thr 260 265 270 Lys Phe 14247 PRT Homo sapiens 14 Met Ala Ser Leu Ser Arg Pro Ser Leu Pro Ser CysLeu Cys Ser Phe 1 5 10 15 Leu Leu Leu Leu Leu Leu Gln Val Ser Ser SerTyr Ala Gly Gln Phe 20 25 30 Arg Val Ile Gly Pro Arg His Pro Ile Arg AlaLeu Val Gly Asp Glu 35 40 45 Val Glu Leu Pro Cys Arg Ile Ser Pro Gly LysAsn Ala Thr Gly Met 50 55 60 Glu Val Gly Trp Tyr Arg Pro Pro Phe Ser ArgVal Val His Leu Tyr 65 70 75 80 Arg Asn Gly Lys Asp Gln Asp Gly Asp GlnAla Pro Glu Tyr Arg Gly 85 90 95 Arg Thr Glu Leu Leu Lys Asp Ala Ile GlyGlu Gly Lys Val Thr Leu 100 105 110 Arg Ile Arg Asn Val Arg Phe Ser AspGlu Gly Gly Phe Thr Cys Phe 115 120 125 Phe Arg Asp His Ser Tyr Gln GluGlu Ala Ala Met Glu Leu Lys Val 130 135 140 Glu Asp Pro Phe Tyr Trp ValSer Pro Gly Val Leu Val Leu Leu Ala 145 150 155 160 Val Leu Pro Val LeuLeu Leu Gln Ile Thr Leu Gly Leu Val Phe Leu 165 170 175 Cys Leu Gln TyrArg Leu Arg Gly Lys Leu Arg Ala Glu Ile Glu Asn 180 185 190 Leu His ArgThr Phe Asp Pro His Phe Leu Arg Val Pro Cys Trp Lys 195 200 205 Ile ThrLeu Phe Val Ile Val Pro Val Leu Gly Pro Leu Val Ala Leu 210 215 220 IleIle Cys Tyr Asn Trp Leu His Arg Arg Leu Ala Gly Gln Phe Leu 225 230 235240 Glu Glu Leu Arg Asn Pro Phe 245 15 18 PRT Rattus norvegicus 15 AlaPro Lys Arg Gly Ser Gly Lys Asp Ser His Thr Arg Thr Thr His 1 5 10 15Tyr Gly 16 23 PRT Homo sapiens 16 Val Leu Gly Gly Gly Cys Ala Leu LeuArg Cys Pro Ala Leu Asp Ser 1 5 10 15 Leu Thr Pro Ala Asn Glu Asp 20 174684 DNA Rattus norvegicus CDS (253)..(3744) 17 attgctcgtc tgggcggcggcggcggctgc agcctgggac agggcgggtg gcacatctcg 60 atcgcgaagg caggagaagcagtctcattg ttccgggagc cgtcgcctct gcaggttctt 120 cggctcggct cggcacgactcggcctgcct ggcccctgcc agtcttgccc aacccccaca 180 accgcccgcg actctgaggagaagcggccc tgcggcggct gtagctgcag catcgtcggc 240 gacccgccag cc atg gaagac ata gac cag tcg tcg ctg gtc tcc tcg tcc 291 Met Glu Asp Ile Asp GlnSer Ser Leu Val Ser Ser Ser 1 5 10 acg gac agc ccg ccc cgg cct ccg cccgcc ttc aag tac cag ttc gtg 339 Thr Asp Ser Pro Pro Arg Pro Pro Pro AlaPhe Lys Tyr Gln Phe Val 15 20 25 acg gag ccc gag gac gag gag gac gag gaggag gag gag gac gag gag 387 Thr Glu Pro Glu Asp Glu Glu Asp Glu Glu GluGlu Glu Asp Glu Glu 30 35 40 45 gag gac gac gag gac cta gag gaa ctg gaggtg ctg gag agg aag ccc 435 Glu Asp Asp Glu Asp Leu Glu Glu Leu Glu ValLeu Glu Arg Lys Pro 50 55 60 gca gcc ggg ctg tcc gca gct gcg gtg ccg cccgcc gcc gcc gcg ccg 483 Ala Ala Gly Leu Ser Ala Ala Ala Val Pro Pro AlaAla Ala Ala Pro 65 70 75 ctg ctg gac ttc agc agc gac tcg gtg ccc ccc gcgccc cgc ggg ccg 531 Leu Leu Asp Phe Ser Ser Asp Ser Val Pro Pro Ala ProArg Gly Pro 80 85 90 ctg ccg gcc gcg ccc cct gcc gct cct gag agg cag ccatcc tgg gaa 579 Leu Pro Ala Ala Pro Pro Ala Ala Pro Glu Arg Gln Pro SerTrp Glu 95 100 105 cgc agc ccc gcg gcg ccc gcg cca tcc ctg ccg ccc gctgcc gca gtc 627 Arg Ser Pro Ala Ala Pro Ala Pro Ser Leu Pro Pro Ala AlaAla Val 110 115 120 125 ctg ccc tcc aag ctc cca gag gac gac gag cct ccggcg agg ccc ccg 675 Leu Pro Ser Lys Leu Pro Glu Asp Asp Glu Pro Pro AlaArg Pro Pro 130 135 140 cct ccg ccg cca gcc ggc gcg agc ccc ctg gcg gagccc gcc gcg ccc 723 Pro Pro Pro Pro Ala Gly Ala Ser Pro Leu Ala Glu ProAla Ala Pro 145 150 155 cct tcc acg ccg gcc gcg ccc aag cgc agg ggc tccggc tca gtg gat 771 Pro Ser Thr Pro Ala Ala Pro Lys Arg Arg Gly Ser GlySer Val Asp 160 165 170 gag acc ctt ttt gct ctt cct gct gca tct gag cctgtg ata ccc tcc 819 Glu Thr Leu Phe Ala Leu Pro Ala Ala Ser Glu Pro ValIle Pro Ser 175 180 185 tct gca gaa aaa att atg gat ttg atg gag cag ccaggt aac act gtt 867 Ser Ala Glu Lys Ile Met Asp Leu Met Glu Gln Pro GlyAsn Thr Val 190 195 200 205 tcg tct ggt caa gag gat ttc cca tct gtc ctgctt gaa act gct gcc 915 Ser Ser Gly Gln Glu Asp Phe Pro Ser Val Leu LeuGlu Thr Ala Ala 210 215 220 tct ctt cct tct cta tct cct ctc tca act gtttct ttt aaa gaa cat 963 Ser Leu Pro Ser Leu Ser Pro Leu Ser Thr Val SerPhe Lys Glu His 225 230 235 gga tac ctt ggt aac tta tca gca gtg tca tcctca gaa gga aca att 1011 Gly Tyr Leu Gly Asn Leu Ser Ala Val Ser Ser SerGlu Gly Thr Ile 240 245 250 gaa gaa act tta aat gaa gct tct aaa gag ttgcca gag agg gca aca 1059 Glu Glu Thr Leu Asn Glu Ala Ser Lys Glu Leu ProGlu Arg Ala Thr 255 260 265 aat cca ttt gta aat aga gat tta gca gaa ttttca gaa tta gaa tat 1107 Asn Pro Phe Val Asn Arg Asp Leu Ala Glu Phe SerGlu Leu Glu Tyr 270 275 280 285 tca gaa atg gga tca tct ttt aaa ggc tcccca aaa gga gag tca gcc 1155 Ser Glu Met Gly Ser Ser Phe Lys Gly Ser ProLys Gly Glu Ser Ala 290 295 300 ata tta gta gaa aac act aag gaa gaa gtaatt gtg agg agt aaa gac 1203 Ile Leu Val Glu Asn Thr Lys Glu Glu Val IleVal Arg Ser Lys Asp 305 310 315 aaa gag gat tta gtt tgt agt gca gcc cttcac agt cca caa gaa tca 1251 Lys Glu Asp Leu Val Cys Ser Ala Ala Leu HisSer Pro Gln Glu Ser 320 325 330 cct gtg ggt aaa gaa gac aga gtt gtg tctcca gaa aag aca atg gac 1299 Pro Val Gly Lys Glu Asp Arg Val Val Ser ProGlu Lys Thr Met Asp 335 340 345 att ttt aat gaa atg cag atg tca gta gtagca cct gtg agg gaa gag 1347 Ile Phe Asn Glu Met Gln Met Ser Val Val AlaPro Val Arg Glu Glu 350 355 360 365 tat gca gac ttt aag cca ttt gaa caagca tgg gaa gtg aaa gat act 1395 Tyr Ala Asp Phe Lys Pro Phe Glu Gln AlaTrp Glu Val Lys Asp Thr 370 375 380 tat gag gga agt agg gat gtg ctg gctgct aga gct aat gtg gaa agt 1443 Tyr Glu Gly Ser Arg Asp Val Leu Ala AlaArg Ala Asn Val Glu Ser 385 390 395 aaa gtg gac aga aaa tgc ttg gaa gatagc ctg gag caa aaa agt ctt 1491 Lys Val Asp Arg Lys Cys Leu Glu Asp SerLeu Glu Gln Lys Ser Leu 400 405 410 ggg aag gat agt gaa ggc aga aat gaggat gct tct ttc ccc agt acc 1539 Gly Lys Asp Ser Glu Gly Arg Asn Glu AspAla Ser Phe Pro Ser Thr 415 420 425 cca gaa cct gtg aag gac agc tcc agagca tat att acc tgt gct tcc 1587 Pro Glu Pro Val Lys Asp Ser Ser Arg AlaTyr Ile Thr Cys Ala Ser 430 435 440 445 ttt acc tca gca acc gaa agc accaca gca aac act ttc cct ttg tta 1635 Phe Thr Ser Ala Thr Glu Ser Thr ThrAla Asn Thr Phe Pro Leu Leu 450 455 460 gaa gat cat act tca gaa aat aaaaca gat gaa aaa aaa ata gaa gaa 1683 Glu Asp His Thr Ser Glu Asn Lys ThrAsp Glu Lys Lys Ile Glu Glu 465 470 475 agg aag gcc caa att ata aca gagaag act agc ccc aaa acg tca aat 1731 Arg Lys Ala Gln Ile Ile Thr Glu LysThr Ser Pro Lys Thr Ser Asn 480 485 490 cct ttc ctt gta gca gta cag gattct gag gca gat tat gtt aca aca 1779 Pro Phe Leu Val Ala Val Gln Asp SerGlu Ala Asp Tyr Val Thr Thr 495 500 505 gat acc tta tca aag gtg act gaggca gca gtg tca aac atg cct gaa 1827 Asp Thr Leu Ser Lys Val Thr Glu AlaAla Val Ser Asn Met Pro Glu 510 515 520 525 ggt ctg acg cca gat tta gttcag gaa gca tgt gaa agt gaa ctg aat 1875 Gly Leu Thr Pro Asp Leu Val GlnGlu Ala Cys Glu Ser Glu Leu Asn 530 535 540 gaa gcc aca ggt aca aag attgct tat gaa aca aaa gtg gac ttg gtc 1923 Glu Ala Thr Gly Thr Lys Ile AlaTyr Glu Thr Lys Val Asp Leu Val 545 550 555 caa aca tca gaa gct ata caagaa tca ctt tac ccc aca gca cag ctt 1971 Gln Thr Ser Glu Ala Ile Gln GluSer Leu Tyr Pro Thr Ala Gln Leu 560 565 570 tgc cca tca ttt gag gaa gctgaa gca act ccg tca cca gtt ttg cct 2019 Cys Pro Ser Phe Glu Glu Ala GluAla Thr Pro Ser Pro Val Leu Pro 575 580 585 gat att gtt atg gaa gca ccatta aat tct ctc ctt cca agc gct ggt 2067 Asp Ile Val Met Glu Ala Pro LeuAsn Ser Leu Leu Pro Ser Ala Gly 590 595 600 605 gct tct gta gtg cag cccagt gta tcc cca ctg gaa gca cct cct cca 2115 Ala Ser Val Val Gln Pro SerVal Ser Pro Leu Glu Ala Pro Pro Pro 610 615 620 gtt agt tat gac agt ataaag ctt gag cct gaa aac ccc cca cca tat 2163 Val Ser Tyr Asp Ser Ile LysLeu Glu Pro Glu Asn Pro Pro Pro Tyr 625 630 635 gaa gaa gcc atg aat gtagca cta aaa gct ttg gga aca aag gaa gga 2211 Glu Glu Ala Met Asn Val AlaLeu Lys Ala Leu Gly Thr Lys Glu Gly 640 645 650 ata aaa gag cct gaa agtttt aat gca gct gtt cag gaa aca gaa gct 2259 Ile Lys Glu Pro Glu Ser PheAsn Ala Ala Val Gln Glu Thr Glu Ala 655 660 665 cct tat ata tcc att gcgtgt gat tta att aaa gaa aca aag ctc tcc 2307 Pro Tyr Ile Ser Ile Ala CysAsp Leu Ile Lys Glu Thr Lys Leu Ser 670 675 680 685 act gag cca agt ccagat ttc tct aat tat tca gaa ata gca aaa ttc 2355 Thr Glu Pro Ser Pro AspPhe Ser Asn Tyr Ser Glu Ile Ala Lys Phe 690 695 700 gag aag tcg gtg cccgaa cac gct gag cta gtg gag gat tcc tca cct 2403 Glu Lys Ser Val Pro GluHis Ala Glu Leu Val Glu Asp Ser Ser Pro 705 710 715 gaa tct gaa cca gttgac tta ttt agt gat gat tcg att cct gaa gtc 2451 Glu Ser Glu Pro Val AspLeu Phe Ser Asp Asp Ser Ile Pro Glu Val 720 725 730 cca caa aca caa gaggag gct gtg atg ctc atg aag gag agt ctc act 2499 Pro Gln Thr Gln Glu GluAla Val Met Leu Met Lys Glu Ser Leu Thr 735 740 745 gaa gtg tct gag acagta gcc cag cac aaa gag gag aga ctt agt gcc 2547 Glu Val Ser Glu Thr ValAla Gln His Lys Glu Glu Arg Leu Ser Ala 750 755 760 765 tca cct cag gagcta gga aag cca tat tta gag tct ttt cag ccc aat 2595 Ser Pro Gln Glu LeuGly Lys Pro Tyr Leu Glu Ser Phe Gln Pro Asn 770 775 780 tta cat agt acaaaa gat gct gca tct aat gac att cca aca ttg acc 2643 Leu His Ser Thr LysAsp Ala Ala Ser Asn Asp Ile Pro Thr Leu Thr 785 790 795 aaa aag gag aaaatt tct ttg caa atg gaa gag ttt aat act gca att 2691 Lys Lys Glu Lys IleSer Leu Gln Met Glu Glu Phe Asn Thr Ala Ile 800 805 810 tat tca aat gatgac tta ctt tct tct aag gaa gac aaa ata aaa gaa 2739 Tyr Ser Asn Asp AspLeu Leu Ser Ser Lys Glu Asp Lys Ile Lys Glu 815 820 825 agt gaa aca ttttca gat tca tct ccg att gag ata ata gat gaa ttt 2787 Ser Glu Thr Phe SerAsp Ser Ser Pro Ile Glu Ile Ile Asp Glu Phe 830 835 840 845 ccc acg tttgtc agt gct aaa gat gat tct cct aaa tta gcc aag gag 2835 Pro Thr Phe ValSer Ala Lys Asp Asp Ser Pro Lys Leu Ala Lys Glu 850 855 860 tac act gatcta gaa gta tcc gac aaa agt gaa att gct aat atc caa 2883 Tyr Thr Asp LeuGlu Val Ser Asp Lys Ser Glu Ile Ala Asn Ile Gln 865 870 875 agc ggg gcagat tca ttg cct tgc tta gaa ttg ccc tgt gac ctt tct 2931 Ser Gly Ala AspSer Leu Pro Cys Leu Glu Leu Pro Cys Asp Leu Ser 880 885 890 ttc aag aatata tat cct aaa gat gaa gta cat gtt tca gat gaa ttc 2979 Phe Lys Asn IleTyr Pro Lys Asp Glu Val His Val Ser Asp Glu Phe 895 900 905 tcc gaa aatagg tcc agt gta tct aag gca tcc ata tcg cct tca aat 3027 Ser Glu Asn ArgSer Ser Val Ser Lys Ala Ser Ile Ser Pro Ser Asn 910 915 920 925 gtc tctgct ttg gaa cct cag aca gaa atg ggc agc ata gtt aaa tcc 3075 Val Ser AlaLeu Glu Pro Gln Thr Glu Met Gly Ser Ile Val Lys Ser 930 935 940 aaa tcactt acg aaa gaa gca gag aaa aaa ctt cct tct gac aca gag 3123 Lys Ser LeuThr Lys Glu Ala Glu Lys Lys Leu Pro Ser Asp Thr Glu 945 950 955 aaa gaggac aga tcc ctg tca gct gta ttg tca gca gag ctg agt aaa 3171 Lys Glu AspArg Ser Leu Ser Ala Val Leu Ser Ala Glu Leu Ser Lys 960 965 970 act tcagtt gtt gac ctc ctc tac tgg aga gac att aag aag act gga 3219 Thr Ser ValVal Asp Leu Leu Tyr Trp Arg Asp Ile Lys Lys Thr Gly 975 980 985 gtg gtgttt ggt gcc agc tta ttc ctg ctg ctg tct ctg aca gtg ttc 3267 Val Val PheGly Ala Ser Leu Phe Leu Leu Leu Ser Leu Thr Val Phe 990 995 1000 1005agc att gtc agt gta acg gcc tac att gcc ttg gcc ctg ctc tcg 3312 Ser IleVal Ser Val Thr Ala Tyr Ile Ala Leu Ala Leu Leu Ser 1010 1015 1020 gtgact atc agc ttt agg ata tat aag ggc gtg atc cag gct atc 3357 Val Thr IleSer Phe Arg Ile Tyr Lys Gly Val Ile Gln Ala Ile 1025 1030 1035 cag aaatca gat gaa ggc cac cca ttc agg gca tat tta gaa tct 3402 Gln Lys Ser AspGlu Gly His Pro Phe Arg Ala Tyr Leu Glu Ser 1040 1045 1050 gaa gtt gctata tca gag gaa ttg gtt cag aaa tac agt aat tct 3447 Glu Val Ala Ile SerGlu Glu Leu Val Gln Lys Tyr Ser Asn Ser 1055 1060 1065 gct ctt ggt catgtg aac agc aca ata aaa gaa ctg agg cgg ctt 3492 Ala Leu Gly His Val AsnSer Thr Ile Lys Glu Leu Arg Arg Leu 1070 1075 1080 ttc tta gtt gat gattta gtt gat tcc ctg aag ttt gca gtg ttg 3537 Phe Leu Val Asp Asp Leu ValAsp Ser Leu Lys Phe Ala Val Leu 1085 1090 1095 atg tgg gtg ttt act tatgtt ggt gcc ttg ttc aat ggt ctg aca 3582 Met Trp Val Phe Thr Tyr Val GlyAla Leu Phe Asn Gly Leu Thr 1100 1105 1110 cta ctg att tta gct ctg atctca ctc ttc agt att cct gtt att 3627 Leu Leu Ile Leu Ala Leu Ile Ser LeuPhe Ser Ile Pro Val Ile 1115 1120 1125 tat gaa cgg cat cag gtg cag atagat cat tat cta gga ctt gca 3672 Tyr Glu Arg His Gln Val Gln Ile Asp HisTyr Leu Gly Leu Ala 1130 1135 1140 aac aag agt gtt aag gat gcc atg gccaaa atc caa gca aaa atc 3717 Asn Lys Ser Val Lys Asp Ala Met Ala Lys IleGln Ala Lys Ile 1145 1150 1155 cct gga ttg aag cgc aaa gca gat tgaaaaagcccca aacagaagtt 3764 Pro Gly Leu Lys Arg Lys Ala Asp 1160catctttaaa ggggacactc acttgattac gggggtggga gggtcagggg tgagcccttg 3824gtggccgtgc ggtttcagct ctttattttt agcagtgcac tgtttgagga aaaattacct 3884gtcttgactt cctgtgttta tcatcttaag tattgtaagc tgctgtgtat ggatctcatt 3944gtagtcacac ttgtcttccc caatgaggcg cctggtgaat aaaggactcg gggaaagctg 4004tgcattgtat ctgctgcagg gtagtctagc tgtatgcaga gagttgtaaa gaaggcaaat 4064ctgggggcag ggaaaaccct tttcacagtg tactgtgttt ggtcagtgta aaactgatgc 4124agatttttct gaaatgaaat gtttagatga gagcatacta ctaaagcaga gtggaaaact 4184ctgtctttat ggtgtgttct aggtgtattg tgaatttact gttatattgc caatataagt 4244aaatatagac ctaatctata tatagtgttt cacaaagctt agatctttaa ccttgcagct 4304gccccacagt gcttgacctc tgagtcattg gttatgcagt gtagtccaag cacataaact 4364aggaagagaa atgtatttgt aggagtgcta cctaccacct gttttcaaga aaatatagaa 4424ctccaacaaa aatatagaat gtcatttcaa agacttactg tatgtatagt taattttgtc 4484acagactctg aaattctatg gactgaattt catgcttcca aatgtttgca gttatcaaac 4544attgttatgc aagaaatcat aaaatgaaga cttataccat tgtggtttaa gccgtactga 4604attatctgtg gaatgcattg tgaactgtaa aagcaaagta tcaataaagc ttatagatct 4664taaaaaaaaa aaaaaaaaaa 4684 18 1163 PRT Rattus norvegicus 18 Met Glu AspIle Asp Gln Ser Ser Leu Val Ser Ser Ser Thr Asp Ser 1 5 10 15 Pro ProArg Pro Pro Pro Ala Phe Lys Tyr Gln Phe Val Thr Glu Pro 20 25 30 Glu AspGlu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Glu Asp Asp 35 40 45 Glu AspLeu Glu Glu Leu Glu Val Leu Glu Arg Lys Pro Ala Ala Gly 50 55 60 Leu SerAla Ala Ala Val Pro Pro Ala Ala Ala Ala Pro Leu Leu Asp 65 70 75 80 PheSer Ser Asp Ser Val Pro Pro Ala Pro Arg Gly Pro Leu Pro Ala 85 90 95 AlaPro Pro Ala Ala Pro Glu Arg Gln Pro Ser Trp Glu Arg Ser Pro 100 105 110Ala Ala Pro Ala Pro Ser Leu Pro Pro Ala Ala Ala Val Leu Pro Ser 115 120125 Lys Leu Pro Glu Asp Asp Glu Pro Pro Ala Arg Pro Pro Pro Pro Pro 130135 140 Pro Ala Gly Ala Ser Pro Leu Ala Glu Pro Ala Ala Pro Pro Ser Thr145 150 155 160 Pro Ala Ala Pro Lys Arg Arg Gly Ser Gly Ser Val Asp GluThr Leu 165 170 175 Phe Ala Leu Pro Ala Ala Ser Glu Pro Val Ile Pro SerSer Ala Glu 180 185 190 Lys Ile Met Asp Leu Met Glu Gln Pro Gly Asn ThrVal Ser Ser Gly 195 200 205 Gln Glu Asp Phe Pro Ser Val Leu Leu Glu ThrAla Ala Ser Leu Pro 210 215 220 Ser Leu Ser Pro Leu Ser Thr Val Ser PheLys Glu His Gly Tyr Leu 225 230 235 240 Gly Asn Leu Ser Ala Val Ser SerSer Glu Gly Thr Ile Glu Glu Thr 245 250 255 Leu Asn Glu Ala Ser Lys GluLeu Pro Glu Arg Ala Thr Asn Pro Phe 260 265 270 Val Asn Arg Asp Leu AlaGlu Phe Ser Glu Leu Glu Tyr Ser Glu Met 275 280 285 Gly Ser Ser Phe LysGly Ser Pro Lys Gly Glu Ser Ala Ile Leu Val 290 295 300 Glu Asn Thr LysGlu Glu Val Ile Val Arg Ser Lys Asp Lys Glu Asp 305 310 315 320 Leu ValCys Ser Ala Ala Leu His Ser Pro Gln Glu Ser Pro Val Gly 325 330 335 LysGlu Asp Arg Val Val Ser Pro Glu Lys Thr Met Asp Ile Phe Asn 340 345 350Glu Met Gln Met Ser Val Val Ala Pro Val Arg Glu Glu Tyr Ala Asp 355 360365 Phe Lys Pro Phe Glu Gln Ala Trp Glu Val Lys Asp Thr Tyr Glu Gly 370375 380 Ser Arg Asp Val Leu Ala Ala Arg Ala Asn Val Glu Ser Lys Val Asp385 390 395 400 Arg Lys Cys Leu Glu Asp Ser Leu Glu Gln Lys Ser Leu GlyLys Asp 405 410 415 Ser Glu Gly Arg Asn Glu Asp Ala Ser Phe Pro Ser ThrPro Glu Pro 420 425 430 Val Lys Asp Ser Ser Arg Ala Tyr Ile Thr Cys AlaSer Phe Thr Ser 435 440 445 Ala Thr Glu Ser Thr Thr Ala Asn Thr Phe ProLeu Leu Glu Asp His 450 455 460 Thr Ser Glu Asn Lys Thr Asp Glu Lys LysIle Glu Glu Arg Lys Ala 465 470 475 480 Gln Ile Ile Thr Glu Lys Thr SerPro Lys Thr Ser Asn Pro Phe Leu 485 490 495 Val Ala Val Gln Asp Ser GluAla Asp Tyr Val Thr Thr Asp Thr Leu 500 505 510 Ser Lys Val Thr Glu AlaAla Val Ser Asn Met Pro Glu Gly Leu Thr 515 520 525 Pro Asp Leu Val GlnGlu Ala Cys Glu Ser Glu Leu Asn Glu Ala Thr 530 535 540 Gly Thr Lys IleAla Tyr Glu Thr Lys Val Asp Leu Val Gln Thr Ser 545 550 555 560 Glu AlaIle Gln Glu Ser Leu Tyr Pro Thr Ala Gln Leu Cys Pro Ser 565 570 575 PheGlu Glu Ala Glu Ala Thr Pro Ser Pro Val Leu Pro Asp Ile Val 580 585 590Met Glu Ala Pro Leu Asn Ser Leu Leu Pro Ser Ala Gly Ala Ser Val 595 600605 Val Gln Pro Ser Val Ser Pro Leu Glu Ala Pro Pro Pro Val Ser Tyr 610615 620 Asp Ser Ile Lys Leu Glu Pro Glu Asn Pro Pro Pro Tyr Glu Glu Ala625 630 635 640 Met Asn Val Ala Leu Lys Ala Leu Gly Thr Lys Glu Gly IleLys Glu 645 650 655 Pro Glu Ser Phe Asn Ala Ala Val Gln Glu Thr Glu AlaPro Tyr Ile 660 665 670 Ser Ile Ala Cys Asp Leu Ile Lys Glu Thr Lys LeuSer Thr Glu Pro 675 680 685 Ser Pro Asp Phe Ser Asn Tyr Ser Glu Ile AlaLys Phe Glu Lys Ser 690 695 700 Val Pro Glu His Ala Glu Leu Val Glu AspSer Ser Pro Glu Ser Glu 705 710 715 720 Pro Val Asp Leu Phe Ser Asp AspSer Ile Pro Glu Val Pro Gln Thr 725 730 735 Gln Glu Glu Ala Val Met LeuMet Lys Glu Ser Leu Thr Glu Val Ser 740 745 750 Glu Thr Val Ala Gln HisLys Glu Glu Arg Leu Ser Ala Ser Pro Gln 755 760 765 Glu Leu Gly Lys ProTyr Leu Glu Ser Phe Gln Pro Asn Leu His Ser 770 775 780 Thr Lys Asp AlaAla Ser Asn Asp Ile Pro Thr Leu Thr Lys Lys Glu 785 790 795 800 Lys IleSer Leu Gln Met Glu Glu Phe Asn Thr Ala Ile Tyr Ser Asn 805 810 815 AspAsp Leu Leu Ser Ser Lys Glu Asp Lys Ile Lys Glu Ser Glu Thr 820 825 830Phe Ser Asp Ser Ser Pro Ile Glu Ile Ile Asp Glu Phe Pro Thr Phe 835 840845 Val Ser Ala Lys Asp Asp Ser Pro Lys Leu Ala Lys Glu Tyr Thr Asp 850855 860 Leu Glu Val Ser Asp Lys Ser Glu Ile Ala Asn Ile Gln Ser Gly Ala865 870 875 880 Asp Ser Leu Pro Cys Leu Glu Leu Pro Cys Asp Leu Ser PheLys Asn 885 890 895 Ile Tyr Pro Lys Asp Glu Val His Val Ser Asp Glu PheSer Glu Asn 900 905 910 Arg Ser Ser Val Ser Lys Ala Ser Ile Ser Pro SerAsn Val Ser Ala 915 920 925 Leu Glu Pro Gln Thr Glu Met Gly Ser Ile ValLys Ser Lys Ser Leu 930 935 940 Thr Lys Glu Ala Glu Lys Lys Leu Pro SerAsp Thr Glu Lys Glu Asp 945 950 955 960 Arg Ser Leu Ser Ala Val Leu SerAla Glu Leu Ser Lys Thr Ser Val 965 970 975 Val Asp Leu Leu Tyr Trp ArgAsp Ile Lys Lys Thr Gly Val Val Phe 980 985 990 Gly Ala Ser Leu Phe LeuLeu Leu Ser Leu Thr Val Phe Ser Ile Val 995 1000 1005 Ser Val Thr AlaTyr Ile Ala Leu Ala Leu Leu Ser Val Thr Ile 1010 1015 1020 Ser Phe ArgIle Tyr Lys Gly Val Ile Gln Ala Ile Gln Lys Ser 1025 1030 1035 Asp GluGly His Pro Phe Arg Ala Tyr Leu Glu Ser Glu Val Ala 1040 1045 1050 IleSer Glu Glu Leu Val Gln Lys Tyr Ser Asn Ser Ala Leu Gly 1055 1060 1065His Val Asn Ser Thr Ile Lys Glu Leu Arg Arg Leu Phe Leu Val 1070 10751080 Asp Asp Leu Val Asp Ser Leu Lys Phe Ala Val Leu Met Trp Val 10851090 1095 Phe Thr Tyr Val Gly Ala Leu Phe Asn Gly Leu Thr Leu Leu Ile1100 1105 1110 Leu Ala Leu Ile Ser Leu Phe Ser Ile Pro Val Ile Tyr GluArg 1115 1120 1125 His Gln Val Gln Ile Asp His Tyr Leu Gly Leu Ala AsnLys Ser 1130 1135 1140 Val Lys Asp Ala Met Ala Lys Ile Gln Ala Lys IlePro Gly Leu 1145 1150 1155 Lys Arg Lys Ala Asp 1160 19 18 PRT Rattusnorvegicus 19 Ser Tyr Asp Ser Ile Lys Leu Glu Pro Glu Asn Pro Pro ProTyr Glu 1 5 10 15 Glu Ala 20 360 PRT Rattus norvegicus 20 Met Glu AspIle Asp Gln Ser Ser Leu Val Ser Ser Ser Thr Asp Ser 1 5 10 15 Pro ProArg Pro Pro Pro Ala Phe Lys Tyr Gln Phe Val Thr Glu Pro 20 25 30 Glu AspGlu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Glu Asp Asp 35 40 45 Glu AspLeu Glu Glu Leu Glu Val Leu Glu Arg Lys Pro Ala Ala Gly 50 55 60 Leu SerAla Ala Ala Val Pro Pro Ala Ala Ala Ala Pro Leu Leu Asp 65 70 75 80 PheSer Ser Asp Ser Val Pro Pro Ala Pro Arg Gly Pro Leu Pro Ala 85 90 95 AlaPro Pro Ala Ala Pro Glu Arg Gln Pro Ser Trp Glu Arg Ser Pro 100 105 110Ala Ala Pro Ala Pro Ser Leu Pro Pro Ala Ala Ala Val Leu Pro Ser 115 120125 Lys Leu Pro Glu Asp Asp Glu Pro Pro Ala Arg Pro Pro Pro Pro Pro 130135 140 Pro Ala Gly Ala Ser Pro Leu Ala Glu Pro Ala Ala Pro Pro Ser Thr145 150 155 160 Pro Ala Ala Pro Lys Arg Arg Gly Ser Gly Ser Val Val ValAsp Leu 165 170 175 Leu Tyr Trp Arg Asp Ile Lys Lys Thr Gly Val Val PheGly Ala Ser 180 185 190 Leu Phe Leu Leu Leu Ser Leu Thr Val Phe Ser IleVal Ser Val Thr 195 200 205 Ala Tyr Ile Ala Leu Ala Leu Leu Ser Val ThrIle Ser Phe Arg Ile 210 215 220 Tyr Lys Gly Val Ile Gln Ala Ile Gln LysSer Asp Glu Gly His Pro 225 230 235 240 Phe Arg Ala Tyr Leu Glu Ser GluVal Ala Ile Ser Glu Glu Leu Val 245 250 255 Gln Lys Tyr Ser Asn Ser AlaLeu Gly His Val Asn Ser Thr Ile Lys 260 265 270 Glu Leu Arg Arg Leu PheLeu Val Asp Asp Leu Val Asp Ser Leu Lys 275 280 285 Phe Ala Val Leu MetTrp Val Phe Thr Tyr Val Gly Ala Leu Phe Asn 290 295 300 Gly Leu Thr LeuLeu Ile Leu Ala Leu Ile Ser Leu Phe Ser Ile Pro 305 310 315 320 Val IleTyr Glu Arg His Gln Val Gln Ile Asp His Tyr Leu Gly Leu 325 330 335 AlaAsn Lys Ser Val Lys Asp Ala Met Ala Lys Ile Gln Ala Lys Ile 340 345 350Pro Gly Leu Lys Arg Lys Ala Asp 355 360 21 199 PRT Rattus norvegicus 21Met Asp Gly Gln Lys Lys His Trp Lys Asp Lys Val Val Asp Leu Leu 1 5 1015 Tyr Trp Arg Asp Ile Lys Lys Thr Gly Val Val Phe Gly Ala Ser Leu 20 2530 Phe Leu Leu Leu Ser Leu Thr Val Phe Ser Ile Val Ser Val Thr Ala 35 4045 Tyr Ile Ala Leu Ala Leu Leu Ser Val Thr Ile Ser Phe Arg Ile Tyr 50 5560 Lys Gly Val Ile Gln Ala Ile Gln Lys Ser Asp Glu Gly His Pro Phe 65 7075 80 Arg Ala Tyr Leu Glu Ser Glu Val Ala Ile Ser Glu Glu Leu Val Gln 8590 95 Lys Tyr Ser Asn Ser Ala Leu Gly His Val Asn Ser Thr Ile Lys Glu100 105 110 Leu Arg Arg Leu Phe Leu Val Asp Asp Leu Val Asp Ser Leu LysPhe 115 120 125 Ala Val Leu Met Trp Val Phe Thr Tyr Val Gly Ala Leu PheAsn Gly 130 135 140 Leu Thr Leu Leu Ile Leu Ala Leu Ile Ser Leu Phe SerIle Pro Val 145 150 155 160 Ile Tyr Glu Arg His Gln Val Gln Ile Asp HisTyr Leu Gly Leu Ala 165 170 175 Asn Lys Ser Val Lys Asp Ala Met Ala LysIle Gln Ala Lys Ile Pro 180 185 190 Gly Leu Lys Arg Lys Ala Asp 195 223579 DNA Homo sapiens CDS (1)..(3579) 22 atg gaa gac ctg gac cag tct cctctg gtc tcg tcc tcg gac agc cca 48 Met Glu Asp Leu Asp Gln Ser Pro LeuVal Ser Ser Ser Asp Ser Pro 1 5 10 15 ccc cgg ccg cag ccc gcg ttc aagtac cag ttc gtg agg gag ccc gag 96 Pro Arg Pro Gln Pro Ala Phe Lys TyrGln Phe Val Arg Glu Pro Glu 20 25 30 gac gag gag gaa gaa gag gag gag gaagag gag gac gag gac gaa gac 144 Asp Glu Glu Glu Glu Glu Glu Glu Glu GluGlu Asp Glu Asp Glu Asp 35 40 45 ctg gag gag ctg gag gtg ctg gag agg aagccc gcc gcc ggg ctg tcc 192 Leu Glu Glu Leu Glu Val Leu Glu Arg Lys ProAla Ala Gly Leu Ser 50 55 60 gcg gcc cca gtg ccc acc gcc cct gcc gcc ggcgcg ccc ctg atg gac 240 Ala Ala Pro Val Pro Thr Ala Pro Ala Ala Gly AlaPro Leu Met Asp 65 70 75 80 ttc gga aat gac ttc gtg ccg ccg gcg ccc cgggga ccc ctg ccg gcc 288 Phe Gly Asn Asp Phe Val Pro Pro Ala Pro Arg GlyPro Leu Pro Ala 85 90 95 gct ccc ccc gtc gcc ccg gag cgg cag ccg tct tgggac ccg agc ccg 336 Ala Pro Pro Val Ala Pro Glu Arg Gln Pro Ser Trp AspPro Ser Pro 100 105 110 gtg tcg tcg acc gtg ccc gcg cca tcc ccg ctg tctgct gcc gca gtc 384 Val Ser Ser Thr Val Pro Ala Pro Ser Pro Leu Ser AlaAla Ala Val 115 120 125 tcg ccc tcc aag ctc cct gag gac gac gag cct ccggcc cgg cct ccc 432 Ser Pro Ser Lys Leu Pro Glu Asp Asp Glu Pro Pro AlaArg Pro Pro 130 135 140 cct cct ccc ccg gcc agc gtg agc ccc cag gca gagccc gtg tgg acc 480 Pro Pro Pro Pro Ala Ser Val Ser Pro Gln Ala Glu ProVal Trp Thr 145 150 155 160 ccg cca gcc ccg gct ccc gcc gcg ccc ccc tccacc ccg gcc gcg ccc 528 Pro Pro Ala Pro Ala Pro Ala Ala Pro Pro Ser ThrPro Ala Ala Pro 165 170 175 aag cgc agg ggc tcc tcg ggc tca gtg gat gagacc ctt ttt gct ctt 576 Lys Arg Arg Gly Ser Ser Gly Ser Val Asp Glu ThrLeu Phe Ala Leu 180 185 190 cct gct gca tct gag cct gtg ata cgc tcc tctgca gaa aat atg gac 624 Pro Ala Ala Ser Glu Pro Val Ile Arg Ser Ser AlaGlu Asn Met Asp 195 200 205 ttg aag gag cag cca ggt aac act att tcg gctggt caa gag gat ttc 672 Leu Lys Glu Gln Pro Gly Asn Thr Ile Ser Ala GlyGln Glu Asp Phe 210 215 220 cca tct gtc ctg ctt gaa act gct gct tct cttcct tct ctg tct cct 720 Pro Ser Val Leu Leu Glu Thr Ala Ala Ser Leu ProSer Leu Ser Pro 225 230 235 240 ctc tca gcc gct tct ttc aaa gaa cat gaatac ctt ggt aat ttg tca 768 Leu Ser Ala Ala Ser Phe Lys Glu His Glu TyrLeu Gly Asn Leu Ser 245 250 255 aca gta tta ccc act gaa gga aca ctt caagaa aat gtc agt gaa gct 816 Thr Val Leu Pro Thr Glu Gly Thr Leu Gln GluAsn Val Ser Glu Ala 260 265 270 tct aaa gag gtc tca gag aag gca aaa actcta ctc ata gat aga gat 864 Ser Lys Glu Val Ser Glu Lys Ala Lys Thr LeuLeu Ile Asp Arg Asp 275 280 285 tta aca gag ttt tca gaa tta gaa tac tcagaa atg gga tca tcg ttc 912 Leu Thr Glu Phe Ser Glu Leu Glu Tyr Ser GluMet Gly Ser Ser Phe 290 295 300 agt gtc tct cca aaa gca gaa tct gcc gtaata gta gca aat cct agg 960 Ser Val Ser Pro Lys Ala Glu Ser Ala Val IleVal Ala Asn Pro Arg 305 310 315 320 gaa gaa ata atc gtg aaa aat aaa gatgaa gaa gag aag tta gtt agt 1008 Glu Glu Ile Ile Val Lys Asn Lys Asp GluGlu Glu Lys Leu Val Ser 325 330 335 aat aac atc ctt cat aat caa caa gagtta cct aca gct ctt act aaa 1056 Asn Asn Ile Leu His Asn Gln Gln Glu LeuPro Thr Ala Leu Thr Lys 340 345 350 ttg gtt aaa gag gat gaa gtt gtg tcttca gaa aaa gca aaa gac agt 1104 Leu Val Lys Glu Asp Glu Val Val Ser SerGlu Lys Ala Lys Asp Ser 355 360 365 ttt aat gaa aag aga gtt gca gtg gaagct cct atg agg gag gaa tat 1152 Phe Asn Glu Lys Arg Val Ala Val Glu AlaPro Met Arg Glu Glu Tyr 370 375 380 gca gac ttc aaa cca ttt gag cga gtatgg gaa gtg aaa gat agt aag 1200 Ala Asp Phe Lys Pro Phe Glu Arg Val TrpGlu Val Lys Asp Ser Lys 385 390 395 400 gaa gat agt gat atg ttg gct gctgga ggt aaa atc gag agc aac ttg 1248 Glu Asp Ser Asp Met Leu Ala Ala GlyGly Lys Ile Glu Ser Asn Leu 405 410 415 gaa agt aaa gtg gat aaa aaa tgtttt gca gat agc ctt gag caa act 1296 Glu Ser Lys Val Asp Lys Lys Cys PheAla Asp Ser Leu Glu Gln Thr 420 425 430 aat cac gaa aaa gat agt gag agtagt aat gat gat act tct ttc ccc 1344 Asn His Glu Lys Asp Ser Glu Ser SerAsn Asp Asp Thr Ser Phe Pro 435 440 445 agt acg cca gaa ggt ata aag gatcgt cca gga gca tat atc aca tgt 1392 Ser Thr Pro Glu Gly Ile Lys Asp ArgPro Gly Ala Tyr Ile Thr Cys 450 455 460 gct ccc ttt aac cca gca gca actgag agc att gca aca aac att ttt 1440 Ala Pro Phe Asn Pro Ala Ala Thr GluSer Ile Ala Thr Asn Ile Phe 465 470 475 480 cct ttg tta gga gat cct acttca gaa aat aag acc gat gaa aaa aaa 1488 Pro Leu Leu Gly Asp Pro Thr SerGlu Asn Lys Thr Asp Glu Lys Lys 485 490 495 ata gaa gaa aag aag gcc caaata gta aca gag aag aat act agc acc 1536 Ile Glu Glu Lys Lys Ala Gln IleVal Thr Glu Lys Asn Thr Ser Thr 500 505 510 aaa aca tca aac cct ttt cttgta gca gca cag gat tct gag aca gat 1584 Lys Thr Ser Asn Pro Phe Leu ValAla Ala Gln Asp Ser Glu Thr Asp 515 520 525 tat gtc aca aca gat aat ttaaca aag gtg act gag gaa gtc gtg gca 1632 Tyr Val Thr Thr Asp Asn Leu ThrLys Val Thr Glu Glu Val Val Ala 530 535 540 aac atg cct gaa ggc ctg actcca gat tta gta cag gaa gca tgt gaa 1680 Asn Met Pro Glu Gly Leu Thr ProAsp Leu Val Gln Glu Ala Cys Glu 545 550 555 560 agt gaa ttg aat gaa gttact ggt aca aag att gct tat gaa aca aaa 1728 Ser Glu Leu Asn Glu Val ThrGly Thr Lys Ile Ala Tyr Glu Thr Lys 565 570 575 atg gac ttg gtt caa acatca gaa gtt atg caa gag tca ctc tat cct 1776 Met Asp Leu Val Gln Thr SerGlu Val Met Gln Glu Ser Leu Tyr Pro 580 585 590 gca gca cag ctt tgc ccatca ttt gaa gag tca gaa gct act cct tca 1824 Ala Ala Gln Leu Cys Pro SerPhe Glu Glu Ser Glu Ala Thr Pro Ser 595 600 605 cca gtt ttg cct gac attgtt atg gaa gca cca ttg aat tct gca gtt 1872 Pro Val Leu Pro Asp Ile ValMet Glu Ala Pro Leu Asn Ser Ala Val 610 615 620 cct agt gct ggt gct tccgtg ata cag ccc agc tca tca cca tta gaa 1920 Pro Ser Ala Gly Ala Ser ValIle Gln Pro Ser Ser Ser Pro Leu Glu 625 630 635 640 gct tct tca gtt aattat gaa agc ata aaa cat gag cct gaa aac ccc 1968 Ala Ser Ser Val Asn TyrGlu Ser Ile Lys His Glu Pro Glu Asn Pro 645 650 655 cca cca tat gaa gaggcc atg agt gta tca cta aaa aaa gta tca gga 2016 Pro Pro Tyr Glu Glu AlaMet Ser Val Ser Leu Lys Lys Val Ser Gly 660 665 670 ata aag gaa gaa attaaa gag cct gaa aat att aat gca gct ctt caa 2064 Ile Lys Glu Glu Ile LysGlu Pro Glu Asn Ile Asn Ala Ala Leu Gln 675 680 685 gaa aca gaa gct ccttat ata tct att gca tgt gat tta att aaa gaa 2112 Glu Thr Glu Ala Pro TyrIle Ser Ile Ala Cys Asp Leu Ile Lys Glu 690 695 700 aca aag ctt tct gctgaa cca gct ccg gat ttc tct gat tat tca gaa 2160 Thr Lys Leu Ser Ala GluPro Ala Pro Asp Phe Ser Asp Tyr Ser Glu 705 710 715 720 atg gca aaa gttgaa cag cca gtg cct gat cat tct gag cta gtt gaa 2208 Met Ala Lys Val GluGln Pro Val Pro Asp His Ser Glu Leu Val Glu 725 730 735 gat tcc tca cctgat tct gaa cca gtt gac tta ttt agt gat gat tca 2256 Asp Ser Ser Pro AspSer Glu Pro Val Asp Leu Phe Ser Asp Asp Ser 740 745 750 ata cct gac gttcca caa aaa caa gat gaa act gtg atg ctt gtg aaa 2304 Ile Pro Asp Val ProGln Lys Gln Asp Glu Thr Val Met Leu Val Lys 755 760 765 gaa agt ctc actgag act tca ttt gag tca atg ata gaa tat gaa aat 2352 Glu Ser Leu Thr GluThr Ser Phe Glu Ser Met Ile Glu Tyr Glu Asn 770 775 780 aag gaa aaa ctcagt gct ttg cca cct gag gga gga aag cca tat ttg 2400 Lys Glu Lys Leu SerAla Leu Pro Pro Glu Gly Gly Lys Pro Tyr Leu 785 790 795 800 gaa tct tttaag ctc agt tta gat aac aca aaa gat acc ctg tta cct 2448 Glu Ser Phe LysLeu Ser Leu Asp Asn Thr Lys Asp Thr Leu Leu Pro 805 810 815 gat gaa gtttca aca ttg agc aaa aag gag aaa att cct ttg cag atg 2496 Asp Glu Val SerThr Leu Ser Lys Lys Glu Lys Ile Pro Leu Gln Met 820 825 830 gag gag ctcagt act gca gtt tat tca aat gat gac tta ttt att tct 2544 Glu Glu Leu SerThr Ala Val Tyr Ser Asn Asp Asp Leu Phe Ile Ser 835 840 845 aag gaa gcacag ata aga gaa act gaa acg ttt tca gat tca tct cca 2592 Lys Glu Ala GlnIle Arg Glu Thr Glu Thr Phe Ser Asp Ser Ser Pro 850 855 860 att gaa attata gat gag ttc cct aca ttg atc agt tct aaa act gat 2640 Ile Glu Ile IleAsp Glu Phe Pro Thr Leu Ile Ser Ser Lys Thr Asp 865 870 875 880 tca ttttct aaa tta gcc agg gaa tat act gac cta gaa gta tcc cac 2688 Ser Phe SerLys Leu Ala Arg Glu Tyr Thr Asp Leu Glu Val Ser His 885 890 895 aaa agtgaa att gct aat gcc ccg gat gga gct ggg tca ttg cct tgc 2736 Lys Ser GluIle Ala Asn Ala Pro Asp Gly Ala Gly Ser Leu Pro Cys 900 905 910 aca gaattg ccc cat gac ctt tct ttg aag aac ata caa ccc aaa gtt 2784 Thr Glu LeuPro His Asp Leu Ser Leu Lys Asn Ile Gln Pro Lys Val 915 920 925 gaa gagaaa atc agt ttc tca gat gac ttt tct aaa aat ggg tct gct 2832 Glu Glu LysIle Ser Phe Ser Asp Asp Phe Ser Lys Asn Gly Ser Ala 930 935 940 aca tcaaag gtg ctc tta ttg cct cca gat gtt tct gct ttg gcc act 2880 Thr Ser LysVal Leu Leu Leu Pro Pro Asp Val Ser Ala Leu Ala Thr 945 950 955 960 caagca gag ata gag agc ata gtt aaa ccc aaa gtt ctt gtg aaa gaa 2928 Gln AlaGlu Ile Glu Ser Ile Val Lys Pro Lys Val Leu Val Lys Glu 965 970 975 gctgag aaa aaa ctt cct tcc gat aca gaa aaa gag gac aga tca cca 2976 Ala GluLys Lys Leu Pro Ser Asp Thr Glu Lys Glu Asp Arg Ser Pro 980 985 990 tctgct ata ttt tca gca gag ctg agt aaa act tca gtt gtt gac ctc 3024 Ser AlaIle Phe Ser Ala Glu Leu Ser Lys Thr Ser Val Val Asp Leu 995 1000 1005ctg tac tgg aga gac att aag aag act gga gtg gtg ttt ggt gcc 3069 Leu TyrTrp Arg Asp Ile Lys Lys Thr Gly Val Val Phe Gly Ala 1010 1015 1020 agccta ttc ctg ctg ctt tca ttg aca gta ttc agc att gtg agc 3114 Ser Leu PheLeu Leu Leu Ser Leu Thr Val Phe Ser Ile Val Ser 1025 1030 1035 gta acagcc tac att gcc ttg gcc ctg ctc tct gtg acc atc agc 3159 Val Thr Ala TyrIle Ala Leu Ala Leu Leu Ser Val Thr Ile Ser 1040 1045 1050 ttt agg atatac aag ggt gtg atc caa gct atc cag aaa tca gat 3204 Phe Arg Ile Tyr LysGly Val Ile Gln Ala Ile Gln Lys Ser Asp 1055 1060 1065 gaa ggc cac ccattc agg gca tat ctg gaa tct gaa gtt gct ata 3249 Glu Gly His Pro Phe ArgAla Tyr Leu Glu Ser Glu Val Ala Ile 1070 1075 1080 tct gag gag ttg gttcag aag tac agt aat tct gct ctt ggt cat 3294 Ser Glu Glu Leu Val Gln LysTyr Ser Asn Ser Ala Leu Gly His 1085 1090 1095 gtg aac tgc acg ata aaggaa ctc agg cgc ctc ttc tta gtt gat 3339 Val Asn Cys Thr Ile Lys Glu LeuArg Arg Leu Phe Leu Val Asp 1100 1105 1110 gat tta gtt gat tct ctg aagttt gca gtg ttg atg tgg gta ttt 3384 Asp Leu Val Asp Ser Leu Lys Phe AlaVal Leu Met Trp Val Phe 1115 1120 1125 acc tat gtt ggt gcc ttg ttt aatggt ctg aca cta ctg att ttg 3429 Thr Tyr Val Gly Ala Leu Phe Asn Gly LeuThr Leu Leu Ile Leu 1130 1135 1140 gct ctc att tca ctc ttc agt gtt cctgtt att tat gaa cgg cat 3474 Ala Leu Ile Ser Leu Phe Ser Val Pro Val IleTyr Glu Arg His 1145 1150 1155 cag gcg cag ata gat cat tat cta gga cttgca aat aag aat gtt 3519 Gln Ala Gln Ile Asp His Tyr Leu Gly Leu Ala AsnLys Asn Val 1160 1165 1170 aaa gat gct atg gct aaa atc caa gca aaa atccct gga ttg aag 3564 Lys Asp Ala Met Ala Lys Ile Gln Ala Lys Ile Pro GlyLeu Lys 1175 1180 1185 cgc aaa gct gaa tga 3579 Arg Lys Ala Glu 1190 231192 PRT Homo sapiens 23 Met Glu Asp Leu Asp Gln Ser Pro Leu Val Ser SerSer Asp Ser Pro 1 5 10 15 Pro Arg Pro Gln Pro Ala Phe Lys Tyr Gln PheVal Arg Glu Pro Glu 20 25 30 Asp Glu Glu Glu Glu Glu Glu Glu Glu Glu GluAsp Glu Asp Glu Asp 35 40 45 Leu Glu Glu Leu Glu Val Leu Glu Arg Lys ProAla Ala Gly Leu Ser 50 55 60 Ala Ala Pro Val Pro Thr Ala Pro Ala Ala GlyAla Pro Leu Met Asp 65 70 75 80 Phe Gly Asn Asp Phe Val Pro Pro Ala ProArg Gly Pro Leu Pro Ala 85 90 95 Ala Pro Pro Val Ala Pro Glu Arg Gln ProSer Trp Asp Pro Ser Pro 100 105 110 Val Ser Ser Thr Val Pro Ala Pro SerPro Leu Ser Ala Ala Ala Val 115 120 125 Ser Pro Ser Lys Leu Pro Glu AspAsp Glu Pro Pro Ala Arg Pro Pro 130 135 140 Pro Pro Pro Pro Ala Ser ValSer Pro Gln Ala Glu Pro Val Trp Thr 145 150 155 160 Pro Pro Ala Pro AlaPro Ala Ala Pro Pro Ser Thr Pro Ala Ala Pro 165 170 175 Lys Arg Arg GlySer Ser Gly Ser Val Asp Glu Thr Leu Phe Ala Leu 180 185 190 Pro Ala AlaSer Glu Pro Val Ile Arg Ser Ser Ala Glu Asn Met Asp 195 200 205 Leu LysGlu Gln Pro Gly Asn Thr Ile Ser Ala Gly Gln Glu Asp Phe 210 215 220 ProSer Val Leu Leu Glu Thr Ala Ala Ser Leu Pro Ser Leu Ser Pro 225 230 235240 Leu Ser Ala Ala Ser Phe Lys Glu His Glu Tyr Leu Gly Asn Leu Ser 245250 255 Thr Val Leu Pro Thr Glu Gly Thr Leu Gln Glu Asn Val Ser Glu Ala260 265 270 Ser Lys Glu Val Ser Glu Lys Ala Lys Thr Leu Leu Ile Asp ArgAsp 275 280 285 Leu Thr Glu Phe Ser Glu Leu Glu Tyr Ser Glu Met Gly SerSer Phe 290 295 300 Ser Val Ser Pro Lys Ala Glu Ser Ala Val Ile Val AlaAsn Pro Arg 305 310 315 320 Glu Glu Ile Ile Val Lys Asn Lys Asp Glu GluGlu Lys Leu Val Ser 325 330 335 Asn Asn Ile Leu His Asn Gln Gln Glu LeuPro Thr Ala Leu Thr Lys 340 345 350 Leu Val Lys Glu Asp Glu Val Val SerSer Glu Lys Ala Lys Asp Ser 355 360 365 Phe Asn Glu Lys Arg Val Ala ValGlu Ala Pro Met Arg Glu Glu Tyr 370 375 380 Ala Asp Phe Lys Pro Phe GluArg Val Trp Glu Val Lys Asp Ser Lys 385 390 395 400 Glu Asp Ser Asp MetLeu Ala Ala Gly Gly Lys Ile Glu Ser Asn Leu 405 410 415 Glu Ser Lys ValAsp Lys Lys Cys Phe Ala Asp Ser Leu Glu Gln Thr 420 425 430 Asn His GluLys Asp Ser Glu Ser Ser Asn Asp Asp Thr Ser Phe Pro 435 440 445 Ser ThrPro Glu Gly Ile Lys Asp Arg Pro Gly Ala Tyr Ile Thr Cys 450 455 460 AlaPro Phe Asn Pro Ala Ala Thr Glu Ser Ile Ala Thr Asn Ile Phe 465 470 475480 Pro Leu Leu Gly Asp Pro Thr Ser Glu Asn Lys Thr Asp Glu Lys Lys 485490 495 Ile Glu Glu Lys Lys Ala Gln Ile Val Thr Glu Lys Asn Thr Ser Thr500 505 510 Lys Thr Ser Asn Pro Phe Leu Val Ala Ala Gln Asp Ser Glu ThrAsp 515 520 525 Tyr Val Thr Thr Asp Asn Leu Thr Lys Val Thr Glu Glu ValVal Ala 530 535 540 Asn Met Pro Glu Gly Leu Thr Pro Asp Leu Val Gln GluAla Cys Glu 545 550 555 560 Ser Glu Leu Asn Glu Val Thr Gly Thr Lys IleAla Tyr Glu Thr Lys 565 570 575 Met Asp Leu Val Gln Thr Ser Glu Val MetGln Glu Ser Leu Tyr Pro 580 585 590 Ala Ala Gln Leu Cys Pro Ser Phe GluGlu Ser Glu Ala Thr Pro Ser 595 600 605 Pro Val Leu Pro Asp Ile Val MetGlu Ala Pro Leu Asn Ser Ala Val 610 615 620 Pro Ser Ala Gly Ala Ser ValIle Gln Pro Ser Ser Ser Pro Leu Glu 625 630 635 640 Ala Ser Ser Val AsnTyr Glu Ser Ile Lys His Glu Pro Glu Asn Pro 645 650 655 Pro Pro Tyr GluGlu Ala Met Ser Val Ser Leu Lys Lys Val Ser Gly 660 665 670 Ile Lys GluGlu Ile Lys Glu Pro Glu Asn Ile Asn Ala Ala Leu Gln 675 680 685 Glu ThrGlu Ala Pro Tyr Ile Ser Ile Ala Cys Asp Leu Ile Lys Glu 690 695 700 ThrLys Leu Ser Ala Glu Pro Ala Pro Asp Phe Ser Asp Tyr Ser Glu 705 710 715720 Met Ala Lys Val Glu Gln Pro Val Pro Asp His Ser Glu Leu Val Glu 725730 735 Asp Ser Ser Pro Asp Ser Glu Pro Val Asp Leu Phe Ser Asp Asp Ser740 745 750 Ile Pro Asp Val Pro Gln Lys Gln Asp Glu Thr Val Met Leu ValLys 755 760 765 Glu Ser Leu Thr Glu Thr Ser Phe Glu Ser Met Ile Glu TyrGlu Asn 770 775 780 Lys Glu Lys Leu Ser Ala Leu Pro Pro Glu Gly Gly LysPro Tyr Leu 785 790 795 800 Glu Ser Phe Lys Leu Ser Leu Asp Asn Thr LysAsp Thr Leu Leu Pro 805 810 815 Asp Glu Val Ser Thr Leu Ser Lys Lys GluLys Ile Pro Leu Gln Met 820 825 830 Glu Glu Leu Ser Thr Ala Val Tyr SerAsn Asp Asp Leu Phe Ile Ser 835 840 845 Lys Glu Ala Gln Ile Arg Glu ThrGlu Thr Phe Ser Asp Ser Ser Pro 850 855 860 Ile Glu Ile Ile Asp Glu PhePro Thr Leu Ile Ser Ser Lys Thr Asp 865 870 875 880 Ser Phe Ser Lys LeuAla Arg Glu Tyr Thr Asp Leu Glu Val Ser His 885 890 895 Lys Ser Glu IleAla Asn Ala Pro Asp Gly Ala Gly Ser Leu Pro Cys 900 905 910 Thr Glu LeuPro His Asp Leu Ser Leu Lys Asn Ile Gln Pro Lys Val 915 920 925 Glu GluLys Ile Ser Phe Ser Asp Asp Phe Ser Lys Asn Gly Ser Ala 930 935 940 ThrSer Lys Val Leu Leu Leu Pro Pro Asp Val Ser Ala Leu Ala Thr 945 950 955960 Gln Ala Glu Ile Glu Ser Ile Val Lys Pro Lys Val Leu Val Lys Glu 965970 975 Ala Glu Lys Lys Leu Pro Ser Asp Thr Glu Lys Glu Asp Arg Ser Pro980 985 990 Ser Ala Ile Phe Ser Ala Glu Leu Ser Lys Thr Ser Val Val AspLeu 995 1000 1005 Leu Tyr Trp Arg Asp Ile Lys Lys Thr Gly Val Val PheGly Ala 1010 1015 1020 Ser Leu Phe Leu Leu Leu Ser Leu Thr Val Phe SerIle Val Ser 1025 1030 1035 Val Thr Ala Tyr Ile Ala Leu Ala Leu Leu SerVal Thr Ile Ser 1040 1045 1050 Phe Arg Ile Tyr Lys Gly Val Ile Gln AlaIle Gln Lys Ser Asp 1055 1060 1065 Glu Gly His Pro Phe Arg Ala Tyr LeuGlu Ser Glu Val Ala Ile 1070 1075 1080 Ser Glu Glu Leu Val Gln Lys TyrSer Asn Ser Ala Leu Gly His 1085 1090 1095 Val Asn Cys Thr Ile Lys GluLeu Arg Arg Leu Phe Leu Val Asp 1100 1105 1110 Asp Leu Val Asp Ser LeuLys Phe Ala Val Leu Met Trp Val Phe 1115 1120 1125 Thr Tyr Val Gly AlaLeu Phe Asn Gly Leu Thr Leu Leu Ile Leu 1130 1135 1140 Ala Leu Ile SerLeu Phe Ser Val Pro Val Ile Tyr Glu Arg His 1145 1150 1155 Gln Ala GlnIle Asp His Tyr Leu Gly Leu Ala Asn Lys Asn Val 1160 1165 1170 Lys AspAla Met Ala Lys Ile Gln Ala Lys Ile Pro Gly Leu Lys 1175 1180 1185 ArgLys Ala Glu 1190 24 373 PRT Homo sapiens 24 Met Glu Asp Leu Asp Gln SerPro Leu Val Ser Ser Ser Asp Ser Pro 1 5 10 15 Pro Arg Pro Gln Pro AlaPhe Lys Tyr Gln Phe Val Arg Glu Pro Glu 20 25 30 Asp Glu Glu Glu Glu GluGlu Glu Glu Glu Glu Asp Glu Asp Glu Asp 35 40 45 Leu Glu Glu Leu Glu ValLeu Glu Arg Lys Pro Ala Ala Gly Leu Ser 50 55 60 Ala Ala Pro Val Pro ThrAla Pro Ala Ala Gly Ala Pro Leu Met Asp 65 70 75 80 Phe Gly Asn Asp PheVal Pro Pro Ala Pro Arg Gly Pro Leu Pro Ala 85 90 95 Ala Pro Pro Val AlaPro Glu Arg Gln Pro Ser Trp Asp Pro Ser Pro 100 105 110 Val Ser Ser ThrVal Pro Ala Pro Ser Pro Leu Ser Ala Ala Ala Val 115 120 125 Ser Pro SerLys Leu Pro Glu Asp Asp Glu Pro Pro Ala Arg Pro Pro 130 135 140 Pro ProPro Pro Ala Ser Val Ser Pro Gln Ala Glu Pro Val Trp Thr 145 150 155 160Pro Pro Ala Pro Ala Pro Ala Ala Pro Pro Ser Thr Pro Ala Ala Pro 165 170175 Lys Arg Arg Gly Ser Ser Gly Ser Val Val Val Asp Leu Leu Tyr Trp 180185 190 Arg Asp Ile Lys Lys Thr Gly Val Val Phe Gly Ala Ser Leu Phe Leu195 200 205 Leu Leu Ser Leu Thr Val Phe Ser Ile Val Ser Val Thr Ala TyrIle 210 215 220 Ala Leu Ala Leu Leu Ser Val Thr Ile Ser Phe Arg Ile TyrLys Gly 225 230 235 240 Val Ile Gln Ala Ile Gln Lys Ser Asp Glu Gly HisPro Phe Arg Ala 245 250 255 Tyr Leu Glu Ser Glu Val Ala Ile Ser Glu GluLeu Val Gln Lys Tyr 260 265 270 Ser Asn Ser Ala Leu Gly His Val Asn CysThr Ile Lys Glu Leu Arg 275 280 285 Arg Leu Phe Leu Val Asp Asp Leu ValAsp Ser Leu Lys Phe Ala Val 290 295 300 Leu Met Trp Val Phe Thr Tyr ValGly Ala Leu Phe Asn Gly Leu Thr 305 310 315 320 Leu Leu Ile Leu Ala LeuIle Ser Leu Phe Ser Val Pro Val Ile Tyr 325 330 335 Glu Arg His Gln AlaGln Ile Asp His Tyr Leu Gly Leu Ala Asn Lys 340 345 350 Asn Val Lys AspAla Met Ala Lys Ile Gln Ala Lys Ile Pro Gly Leu 355 360 365 Lys Arg LysAla Glu 370 25 199 PRT Homo sapiens 25 Met Asp Gly Gln Lys Lys Asn TrpLys Asp Lys Val Val Asp Leu Leu 1 5 10 15 Tyr Trp Arg Asp Ile Lys LysThr Gly Val Val Phe Gly Ala Ser Leu 20 25 30 Phe Leu Leu Leu Ser Leu ThrVal Phe Ser Ile Val Ser Val Thr Ala 35 40 45 Tyr Ile Ala Leu Ala Leu LeuSer Val Thr Ile Ser Phe Arg Ile Tyr 50 55 60 Lys Gly Val Ile Gln Ala IleGln Lys Ser Asp Glu Gly His Pro Phe 65 70 75 80 Arg Ala Tyr Leu Glu SerGlu Val Ala Ile Ser Glu Glu Leu Val Gln 85 90 95 Lys Tyr Ser Asn Ser AlaLeu Gly His Val Asn Cys Thr Ile Lys Glu 100 105 110 Leu Arg Arg Leu PheLeu Val Asp Asp Leu Val Asp Ser Leu Lys Phe 115 120 125 Ala Val Leu MetTrp Val Phe Thr Tyr Val Gly Ala Leu Phe Asn Gly 130 135 140 Leu Thr LeuLeu Ile Leu Ala Leu Ile Ser Leu Phe Ser Val Pro Val 145 150 155 160 IleTyr Glu Arg His Gln Ala Gln Ile Asp His Tyr Leu Gly Leu Ala 165 170 175Asn Lys Asn Val Lys Asp Ala Met Ala Lys Ile Gln Ala Lys Ile Pro 180 185190 Gly Leu Lys Arg Lys Ala Glu 195 26 473 PRT Homo sapiens 26 Met LysArg Ala Ser Ala Gly Gly Ser Arg Leu Leu Ala Trp Val Leu 1 5 10 15 TrpLeu Gln Ala Trp Gln Val Ala Ala Pro Cys Pro Gly Ala Cys Val 20 25 30 CysTyr Asn Glu Pro Lys Val Thr Thr Ser Cys Pro Gln Gln Gly Leu 35 40 45 GlnAla Val Pro Val Gly Ile Pro Ala Ala Ser Gln Arg Ile Phe Leu 50 55 60 HisGly Asn Arg Ile Ser His Val Pro Ala Ala Ser Phe Arg Ala Cys 65 70 75 80Arg Asn Leu Thr Ile Leu Trp Leu His Ser Asn Val Leu Ala Arg Ile 85 90 95Asp Ala Ala Ala Phe Thr Gly Leu Ala Leu Leu Glu Gln Leu Asp Leu 100 105110 Ser Asp Asn Ala Gln Leu Arg Ser Val Asp Pro Ala Thr Phe His Gly 115120 125 Leu Gly Arg Leu His Thr Leu His Leu Asp Arg Cys Gly Leu Gln Glu130 135 140 Leu Gly Pro Gly Leu Phe Arg Gly Leu Ala Ala Leu Gln Tyr LeuTyr 145 150 155 160 Leu Gln Asp Asn Ala Leu Gln Ala Leu Pro Asp Asp ThrPhe Arg Asp 165 170 175 Leu Gly Asn Leu Thr His Leu Phe Leu His Gly AsnArg Ile Ser Ser 180 185 190 Val Pro Glu Arg Ala Phe Arg Gly Leu His SerLeu Asp Arg Leu Leu 195 200 205 Leu His Gln Asn Arg Val Ala His Val HisPro His Ala Phe Arg Asp 210 215 220 Leu Gly Arg Leu Met Thr Leu Tyr LeuPhe Ala Asn Asn Leu Ser Ala 225 230 235 240 Leu Pro Thr Glu Ala Leu AlaPro Leu Arg Ala Leu Gln Tyr Leu Arg 245 250 255 Leu Asn Asp Asn Pro TrpVal Cys Asp Cys Arg Ala Arg Pro Leu Trp 260 265 270 Ala Trp Leu Gln LysPhe Arg Gly Ser Ser Ser Glu Val Pro Cys Ser 275 280 285 Leu Pro Gln ArgLeu Ala Gly Arg Asp Leu Lys Arg Leu Ala Ala Asn 290 295 300 Asp Leu GlnGly Cys Ala Val Ala Thr Gly Pro Tyr His Pro Ile Trp 305 310 315 320 ThrGly Arg Ala Thr Asp Glu Glu Pro Leu Gly Leu Pro Lys Cys Cys 325 330 335Gln Pro Asp Ala Ala Asp Lys Ala Ser Val Leu Glu Pro Gly Arg Pro 340 345350 Ala Ser Ala Gly Asn Ala Leu Lys Gly Arg Val Pro Pro Gly Asp Ser 355360 365 Pro Pro Gly Asn Gly Ser Gly Pro Arg His Ile Asn Asp Ser Pro Phe370 375 380 Gly Thr Leu Pro Gly Ser Ala Glu Pro Pro Leu Thr Ala Val ArgPro 385 390 395 400 Glu Gly Ser Glu Pro Pro Gly Phe Pro Thr Ser Gly ProArg Arg Arg 405 410 415 Pro Gly Cys Ser Arg Lys Asn Arg Thr Arg Ser HisCys Arg Leu Gly 420 425 430 Gln Ala Gly Ser Gly Gly Gly Gly Thr Gly AspSer Glu Gly Ser Gly 435 440 445 Ala Leu Pro Ser Leu Thr Cys Ser Leu ThrPro Leu Gly Leu Ala Leu 450 455 460 Val Leu Trp Thr Val Leu Gly Pro Cys465 470 27 473 PRT Mus musculus 27 Met Lys Arg Ala Ser Ser Gly Gly SerArg Leu Leu Ala Trp Val Leu 1 5 10 15 Trp Leu Gln Ala Trp Arg Val AlaThr Pro Cys Pro Gly Ala Cys Val 20 25 30 Cys Tyr Asn Glu Pro Lys Val ThrThr Ser Cys Pro Gln Gln Gly Leu 35 40 45 Gln Ala Val Pro Thr Gly Ile ProAla Ser Ser Gln Arg Ile Phe Leu 50 55 60 His Gly Asn Arg Ile Ser His ValPro Ala Ala Ser Phe Gln Ser Cys 65 70 75 80 Arg Asn Leu Thr Ile Leu TrpLeu His Ser Asn Ala Leu Ala Arg Ile 85 90 95 Asp Ala Ala Ala Phe Thr GlyLeu Thr Leu Leu Glu Gln Leu Asp Leu 100 105 110 Ser Asp Asn Ala Gln LeuHis Val Val Asp Pro Thr Thr Phe His Gly 115 120 125 Leu Gly His Leu HisThr Leu His Leu Asp Arg Cys Gly Leu Arg Glu 130 135 140 Leu Gly Pro GlyLeu Phe Arg Gly Leu Ala Ala Leu Gln Tyr Leu Tyr 145 150 155 160 Leu GlnAsp Asn Asn Leu Gln Ala Leu Pro Asp Asn Thr Phe Arg Asp 165 170 175 LeuGly Asn Leu Thr His Leu Phe Leu His Gly Asn Arg Ile Pro Ser 180 185 190Val Pro Glu His Ala Phe Arg Gly Leu His Ser Leu Asp Arg Leu Leu 195 200205 Leu His Gln Asn His Val Ala Arg Val His Pro His Ala Phe Arg Asp 210215 220 Leu Gly Arg Leu Met Thr Leu Tyr Leu Phe Ala Asn Asn Leu Ser Met225 230 235 240 Leu Pro Ala Glu Val Leu Met Pro Leu Arg Ser Leu Gln TyrLeu Arg 245 250 255 Leu Asn Asp Asn Pro Trp Val Cys Asp Cys Arg Ala ArgPro Leu Trp 260 265 270 Ala Trp Leu Gln Lys Phe Arg Gly Ser Ser Ser GluVal Pro Cys Asn 275 280 285 Leu Pro Gln Arg Leu Ala Asp Arg Asp Leu LysArg Leu Ala Ala Ser 290 295 300 Asp Leu Glu Gly Cys Ala Val Ala Ser GlyPro Phe Arg Pro Ile Gln 305 310 315 320 Thr Ser Gln Leu Thr Asp Glu GluLeu Leu Ser Leu Pro Lys Cys Cys 325 330 335 Gln Pro Asp Ala Ala Asp LysAla Ser Val Leu Glu Pro Gly Arg Pro 340 345 350 Ala Ser Ala Gly Asn AlaLeu Lys Gly Arg Val Pro Pro Gly Asp Thr 355 360 365 Pro Pro Gly Asn GlySer Gly Pro Arg His Ile Asn Asp Ser Pro Phe 370 375 380 Gly Thr Leu ProSer Ser Ala Glu Pro Pro Leu Thr Ala Leu Arg Pro 385 390 395 400 Gly GlySer Glu Pro Pro Gly Leu Pro Thr Thr Gly Pro Arg Arg Arg 405 410 415 ProGly Cys Ser Arg Lys Asn Arg Thr Arg Ser His Cys Arg Leu Gly 420 425 430Gln Ala Gly Ser Gly Ala Ser Gly Thr Gly Asp Ala Glu Gly Ser Gly 435 440445 Ala Leu Pro Ala Leu Ala Cys Ser Leu Ala Pro Leu Gly Leu Ala Leu 450455 460 Val Leu Trp Thr Val Leu Gly Pro Cys 465 470 28 15 PRT ArtificialSequence synthetic 28 Ser Gly Val Pro Ser Asn Leu Pro Gln Arg Leu AlaGly Arg Asp 1 5 10 15 29 15 PRT Artificial Sequence synthetic 29 Thr ArgSer His Cys Arg Leu Gly Gln Ala Gly Ser Gly Ser Ser 1 5 10 15

What is claimed is:
 1. A method for promoting nerve regeneration or forconferring neuroprotection and preventing or inhibiting neuronaldegeneration in the central nervous system or peripheral nervous systemfor ameliorating the effects of injury or disease, comprisingadministering to an individual in need thereof at least one ingredientselected from the group consisting of: (a) NS-specific activated Tcells; (b) a NS-specific antigen or an analog thereof; (c) a peptidederived from an NS-specific antigen or from an analog thereof, or ananalog or derivative of said peptide; (d) a nucleotide sequence encodingan NS-specific antigen or an analog thereof; (e) a nucleotide sequenceencoding a peptide derived from an NS-specific antigen or from an analogthereof, or an analog of said peptide; or (f) any combination of(a)-(e).
 2. The method according to claim 1 wherein the injury isselected from the group consisting of spinal cord injury, blunt trauma,penetrating trauma, hemorrhagic stroke, ischemic stroke or damagescaused by surgery such as tumor excision.
 3. The method according toclaim 1 wherein the disease is not an autoimmune disease or a neoplasm.4. The method according to claim 1 wherein the disease results in adegenerative process occurring in either gray or white matter or both.5. The method according to claim 4 wherein said disease is selected fromthe group consisting of diabetic neuropathy, senile dementia,Alzheimer's disease, Parkinson's disease, facial nerve (Bell's) palsy,glaucoma, Huntington's chorea, amyotrophic lateral sclerosis,non-arteritic optic neuropathy, and vitamin deficiency.
 6. The methodaccording to claim 4 wherein said disease is selected from the groupconsisting of intervertebral disc herniation, prion diseases such asCreutzfeldt-Jakob disease, carpal tunnel syndrome, peripheralneuropathies associated with various diseases, including but not limitedto, uremia, porphyria, hypoglycemia, Sjorgren Larsson syndrome, acutesensory neuropathy, chronic ataxic neuropathy, biliary cirrhosis,primary amyloidosis, obstructive lung diseases, acromegaly,malabsorption syndromes, polycythemia vera, IgA- and IgG gamma-pathies,complications of various drugs (e.g., metronidazole) and toxins (e.g.,alcohol or organophosphates), Charcot-Marie-Tooth disease, ataxiatelangectasia, Friedreich's ataxia, amyloid polyneuropathies,adrenomyeloneuropathy, Giant axonal neuropathy, Refsum's disease,Fabry's disease, and lipoproteinemia.
 7. The method according to claim 1which comprises administering to the individual in need NS-specificactivated T cells.
 8. The method according to claim 7 wherein saidNS-specific activated T cells are selected from the group consisting ofautologous T cells, semi-allogeneic T cells or allogeneic T cells fromrelated donors, or from donors who are HLA-matched or HLA-partiallymatched, or from unrelated donors.
 9. The method according to claim 8wherein said T cells are autologous T cells.
 10. The method according toclaim 8 wherein said T cells are semi-allogeneic T cells.
 11. The methodaccording to claim 9 or 10 wherein said autologous or semi-allogeneic Tcells have been sensitized to an NS-specific antigen or an analogthereof.
 12. The method according to claim 11 wherein the NS-specificantigen is selected from the group consisting of myelin basic protein(MBP), myelin oligodendrocyte glycoprotein (MOG), proteolipid protein(PLP), myelin-associated glycoprotein (MAG), S-100, β-amyloid, Thy-1,P0, P2, and a neurotransmitter receptor.
 13. The method according toclaim 12 wherein the NS-specific antigen is MBP.
 14. The methodaccording to claim 11 wherein the NS-specific antigen is selected fromthe group consisting of Nogo-A, Nogo-B, Nogo-C, and Nogo receptor. 15.The method according to claim 9 or 10 wherein said autologous orsemi-allogeneic T cells have been sensitized to a peptide derived froman NS-specific antigen or from an analog thereof, or to an analog orderivative of said peptide.
 16. The method according to claim 15 whereinsaid peptide derived from an NS-specific antigen is an immunogenicepitope or a cryptic epitope of said antigen.
 17. The method accordingto claim 16 wherein said peptide is an immunogenic epitope or a crypticepitope derived from MBP.
 18. The method according to claim 17 whereinsaid peptide corresponds to a peptide selected from the sequencesconsisting of the sequences p11-30, p51-70, p87-99, p91-110, p131-150,and p151-170 of MBP.
 19. The method according to claim 18 wherein saidpeptide corresponds to the sequence p51-70 of MBP.
 20. The methodaccording to claim 16 wherein said peptide is an immunogenic epitope ora cryptic epitope derived from MOG.
 21. The method according to claim 20wherein said @ peptide corresponds to the sequence p35-55 of MOG. 22.The method according to claim 16 wherein said peptide is an immunogenicepitope or a cryptic epitope derived from Nogo.
 23. The method accordingto claim 22 wherein said peptide is the Nogo-A p472 peptide (SEQ IDNO:19).
 24. The method according to claim 16 wherein said peptide is animmunogenic epitope or a cryptic epitope derived from Nogo receptor. 25.The method according to claim 9 or 10 wherein said autologous orsemi-allogeneic T cells have been stored for future use.
 26. The methodaccording to claim 1 which comprises administering to the individual inneed a NS-specific antigen or an analog thereof.
 27. The methodaccording to claim 26 wherein the NS-specific antigen is selected fromthe group consisting of myelin basic protein (MBP), myelinoligodendrocyte glycoprotein (MOG), proteolipid protein (PLP),myelin-associated glycoprotein (MAG), S-100, β-amyloid, Thy-1, P0, P2,and a neurotransmitter receptor.
 28. The method according to claim 27wherein the NS-specific antigen is MBP.
 29. The method according toclaim 28 wherein the MBP is administered orally.
 30. The methodaccording to claim 23 wherein the NS-specific antigen is selected fromthe group consisting of Nogo-A, Nogo-B, Nogo-C, and Nogo receptor. 31.The method according to claim 1 which comprises administering to theindividual in need a peptide derived from an NS-specific antigen or froman analog thereof, or an analog or derivative of said peptide.
 32. Themethod according to claim 31 wherein said peptide derived from anNS-specific antigen is an immunogenic epitope or a cryptic epitope ofsaid antigen.
 33. The method according to claim 32 wherein said peptideis an immunogenic epitope or a cryptic epitope derived from MBP.
 34. Themethod according to claim 33 wherein said peptide corresponds to apeptide selected from the sequences consisting of the sequences p11-30,p51-70, p87-99, p91-110, p131-150, and p151-170 of MBP.
 35. The methodaccording to claim 34 wherein said peptide corresponds to the sequencep51-70 of NBP.
 36. The method according to claim 32 wherein said peptideis an immunogenic epitope or a cryptic epitope derived from MOG.
 37. Themethod according to claim 36 wherein said peptide corresponds to thesequence p35-55 of MOG.
 38. The method according to claim 32 whereinsaid peptide is an immunogenic epitope or a cryptic epitope derived fromNogo.
 39. The method according to claim 38 wherein said peptide is theNogo-A p472 peptide (SEQ ID NO:19).
 40. The method according to claim 32wherein said peptide is an immunogenic epitope or a cryptic epitopederived from Nogo receptor.
 41. The method according to claim 1 whereinsaid NS-specific antigen or a peptide derived therefrom is administeredintravenously, intrathecally, intramuscularly, intradermally, topically,subcutaneously, or mucosally.
 42. The method according to claim 41wherein said mucosal administration is selected from the groupconsisting of oral, intranasal, buccal, vaginal and rectaladministration.
 43. The method according to claim 42 wherein saidNS-specific antigen or peptide derived therefrom is administered orallyand the individual is actively immunized to build up a critical T cellresponse.
 44. A method for preventing or inhibiting neuronaldegeneration in the central nervous system or peripheral nervous systemcomprising administering to an individual in need thereof an effectiveamount of a composition for up-regulating B7.2 co-stimulatory moleculeor genetically manipulating B7.2 co-stimulatory molecule in saidindividual.